Human tissue factor antibody and uses thereof

ABSTRACT

The invention relates to a humanized form of an antibody capable of preventing tissue factor (coagulation factor F3) signaling but which does not interfere with Factor VII binding or FX binding to tissue factor and does not prolong coagulation time. The antibody of the invention is useful in treating conditions, such as tumor progression, in which the associated cells express tissue factor and tissue factor signaling occurs.

PRIOR APPLICATION

This application claims priority to U.S. Application No. 61/452,674,filed Mar. 15, 2011, which is entirely incorporated herein by reference.

BACKGROUND

1. Field of the Invention

The invention relates to human adapted antibodies which bind humantissue factor, an antigen present on extra vascular tissues includingtumor cells, which antibodies do not inhibit tissue factor mediatedblood coagulation. The invention also relates to methods of using theantibody to treat conditions such as cancer that are associated with thepresence and receptor functions of human tissue factor.

2. Discussion of the Field

Tissue Factor (TF), also known as coagulation factor III (F3), tissuethromboplastin, or CD142 is a transmembrane glycoprotein having a 219amino acid extracellular domain comprising two fibronectin type IIIdomains and a short intracellular domain with one serine residue capableof being phosphorylated. TF is the cellular receptor for FVII/FVIIa.

TF exhibits a tissue-specific distribution with high levels in thenormal brain, lung and placenta and low levels in the spleen, thymus,skeletal muscle and liver in the form of a cellular receptor. It is alsofound in cell-derived microparticles and as an alternatively splicedsoluble form. In addition to the expression in normal tissue, TF hasbeen reported to be over-expressed in most major tumor types and in manytumor-derived cell lines (Ruf W J Thromb Haemost. 5:1584-1587, 2007;Milsom et al., Arterioscler Thromb Vasc Biol. 29: 2005-2014, 2009).

Coagulation of serum proteins in response to injury is an importantphysiological response to injury. Exposure of the blood to proteinsincluding collagen (intrinsic pathway) and tissue factor (extrinsicpathway) initiates changes to blood platelets and the plasma proteinfibrinogen, a clotting factor. Following damage to a blood vessel,factor VII (FVII) leaves the circulation and comes into contact withtissue factor (TF) expressed on tissue-factor-bearing cells (stromalfibroblasts and leukocytes), forming an activated TF-FVIIa complex.TF-FVIIa activates factor IX (FIX) and factor X (FX). FVII can beallosterically activated by TF and activated by thrombin, FXIa, plasmin,FXII and FXa. TF-FVIIa forms a ternary complex with FXa.

Tissue factor (TF) expression by nonvascular cells plays an essentialrole in hemostasis by activating blood coagulation. TF is furtherassociated with processes distinct from hemostasis and directly relatedto functions at the surface of cells on which it is expressed.TF-dependent assembly of coagulation proteases on vascular andnonvascular cells activates protease activated receptors (PARs) whichare G-protein-coupled receptors. Thus, the TF:VIIa complex is capable ofinducing cell signaling, through PARs, primarily PAR2 (Camerer et al.,Proc. Natl. Acad. Sci. USA 97:5255-5260, 2000; Riewald & Ruf, Proc.Natl. Acad. Sci. USA 98:7742-7747, 2001; Ruf et al, J Thromb Haemost 1:1495-4503, 2003; Chen et al., Thromb Haemost 86: 334-45, 2001)contributing to tumorigenesis, angiogenesis, tumor progression, andmetastasis.

The ternary complex TF/FVIIa/FXa is formed directly by the TF:VIIacomplex acting on FX or indirectly after TF:VIIa cleavage of FIX to FIXawhich can cleave FX to FXa. The TF/FVIIa/FXa complex formation mayresult in signaling or activate other receptors such as PAR1-4.TF/FVIIa/FXa complex formation leads to the induction of Interleukin-8(IL-8), which can stimulate tumor cell migration (Hjortor et al., Blood103:3029-3037, 2004). Both PAR1 and PAR2 are involved in tumormetastasis (Shi et al., Mol Cancer Res. 2:395-402, 2004), however, theactivated binary and ternary complexes, TF-VIIa and TF-VIIa-FXa, areactivators of PAR2 which also leads to cell signaling (Rao & Pendurthi,Arterioscler. Thromb. Vasc. Biol. 25:47-56, 2005). Therefore, it was ofinterest to determine whether the oncogenic role of tissue factor couldbe separated from the procoagulant role, which had also long beensuspected to be involved in tumor migration, extravasation, andmetastatic mechanisms.

Monoclonal antibodies such as those described by Morrisey (1988, ThrombRes 52(3): 247-261; U.S. Pat. No. 5,223,427) and Magdolen (1996 BiolChem 379: 157-165) to tissue factor have been used to explore functionaland immunological aspects of the ligand binding sites. Monoclonalantibodies capable of binding tissue factor can be used to blockthrombotic events by interfering with the ability of TF to form ormaintain the TF-VIIa complex or by blocking the ability of the complexto activate FX. Antibodies that bind to tissue factor and do not blockcoagulation are also known. Factor VIIa initiated TF signaling blockingbut not coagulation blocking antibodies such as the antibody 10H10 havealso been described (Ahamed et al. 2006 Proc Natl Acad Sci USA 103 (38):13932-13937) and such antibodies have provided the opportunity to studythe role and utility of an agent with such activity in the treatment ofsolid tumors (Versteeg, et al 2008 Blood 111(1): 190-199). Ruf et al, inpublished application WO2007056352A3 discloses methods and compositionsfor inhibiting tissue factor signaling without interfering withhemostatis in a patient.

As cancer progression is a multifaceted process, a therapeutic candidatewhich is a TF binding antibody capable of blockade of oncogenic,metastatic, angiogenic, and anti-apoptotic functions on tumor cellswhile not interfering with hemostasis in a patient would be desirable.

SUMMARY OF THE INVENTION

The present invention provides a human adapted anti-human tissue factorspecific antibody for use as a human therapeutic which retains thebinding epitope of the murine antibody 10H10, which antibody does notcompete with tissue factor for FVIIa binding and therefore does notsubstantially block the procoagulant, amidolytic activity of the TF-VIIacomplex but which does block TF-VIIa mediated signaling and downstreamoncogenic effects such as cytokine IL-8 release.

The human adapted antibody of the invention is constructed of human IgGvariable domain frameworks in combination with CDR variant residues asdetermined by referring to the sequence of the 10H10 murine antibody CDRsequences and as represented as SEQ ID NO: 6-11 and 27. Human frameworksFR1 and FR2 and FR3, combined with the CDRs and CDR variants, with FR4,are provided which allow the assembly of antibody binding domains withthe immunospecificity of the murine antibody 10H10. In one embodiment ofthe invention, the six CDR sequences represented by SEQ ID NO: 6-11 oras the group represented by SEQ ID NO: 6, 8-11, and 27 are combined withhuman germline FRs, defined as the non-CDR positions of a human IgGvariable domain, selected so that the binding affinity of 10H10 forhuman TF is retained. In one aspect, the human HC variable region FRsare derived from an IGHV gene family 1, 3 or 5 member as represented bythe IMGT database. In one aspect, the human LC variable region FRs arederived from a human IGKV gene family 2 or 4 member. In one embodiment,antibody Fv (HC variable region paired with a LC variable region)comprise an HC variable domain selected from SEQ ID NO: 12-21 and a LCvariable domain selected from SEQ ID NO: 22-26.

In a particular embodiment, the human FRs forming an antibody Fv (HCvariable region paired with a LC variable region) comprise IGHV5 andIGKV2 FRs. The antibody of the invention comprises an HC variable domainhaving the H-CDR3 of SEQ ID NO: 8; an H-CDR1 having a sequence selectedfrom SEQ ID NO: 6, and 62-83; an H-CDR2 having a sequence selected fromSEQ ID NO: 7, 27, and 84-107; and an HC FR4 region, optionally, selectedfrom IGVJ4 (SEQ ID NO: 60) or a variant thereof. The antibodies of theinvention further comprise those having an LC variable domain having anL-CDR1 having a sequence selected from SEQ ID NO: 9, 108-116; an L-CDR2having a sequence selected from SEQ ID NO: 10 and 117-120; and an L-CDR3having a sequence selected from SEQ ID NO: 11 and 121-128; and a LC FR4region, optionally, selected from IGKJ2 (SEQ ID NO: 61) or a variantthereof. In a specific embodiment, the human framework sequences arederived from IGHV5_a and the created variable domain comprises asequence selected from SEQ ID NO: 19, 129-155. In another embodiment,the human framework sequences are derived from IGKV2D40_O1 and thecreated variable domain comprises a sequence selected from SEQ ID NO:23, 156-163.

The antibodies of the present invention can be represented in one formas antibodies having a binding domain derived from IGHV5_a frameworks,defined as non-CDR positions, an H-CDR3 having the sequence SGYYGNSGFAY(SEQ ID NO: 8), wherein the sequences at the H-CDR-1 positions is givenby the formula:

H-CDR1 (SEQ ID NO: 83) GYTFX₁X₂X₃WIE (I)

where X1 is selected is selected from A, D, G, I, L, N, P, R, S, T, V,and Y; X2 is selected from A, P, S, and T and X3 is selected from F, H,and Y; or the sequence may be GFTFITYWIA (SEQ ID NO: 81); and thesequence at the H-CDR2 position is given by the formula:

H-CDR2 (SEQ ID NO: 107) DIX₁PGX₂GX₃TX₄ (II)

where X1 is selected from 1 and L, X2 is selected from S and T, X3 isselected from A, F, H, and w; and X4 is selected from D, H, I, L, and N;except in H189 where H-CDR2 is DILPASSSTN (SEQ ID NO: 105).

The antibodies of the invention are represented as antibodies having abinding domain derived from IGKV2D40_O1 frameworks, defined as non-CDRpositions, and wherein the sequences at the L-CDR-1 and/or LCDR-2, andL-CDR3 have the sequences given by the formulas:

L-CDR1 (SEQ ID NO: 116) KSSQSLLX₁X₂X₃ X₄Q X₅NYLT (III)

where X1 is selected from F, P, S, T, W, and Y; X2 is selected from F,S, T, R, and V; X3 is selected from A, G, P, S, W, Y, AND V; X4 isselected from G, N, and T; X5 is selected from K, R, and S;

L-CDR2 (SEQ ID NO: 120) X₁ASTRX₂S (IV)

where X1 is selected from H and W; X2 is selected from D, E and S;

L-CDR3 (SEQ ID NO: 128) QNDX₁X₂X₃PX₄T (V)

where X1 is selected from D, F, and L; X2 is selected from S, T, and Y;X3 is selected from W, and Y; X4 is selected from L, and M.

Thus, the antibody heavy chain and light chain CDR residues aresubstantially modified from the murine CDRs of 10H10. For instance, inaccordance with the description set forth above, the antibody heavychain can be only 70% (3/10 residues altered in CDR1), and 60% (4/10residues altered in CDR2) similar to the murine CDRs of 10H10 (CDR 3 isunchanged). The light chain CDR residues are only 71% (5/17 changed)),(71%) (2/7 changed), or 55% (4/9 changed) similar to the murine CDRs of10H10.

The invention further provides human adapted antibodies that compete forbinding to human tissue factor and thus bind to substantially the sameepitope on human TF-ECD as the murine 10H10 antibody. The inventionfurther provides methods of using such antibodies to treat a humansubject suffering from a condition in which TF-expression and localbioactivity resulting from the TF-expression is directly or indirectlyrelated to the condition to be treated.

The invention further provides methods for preparing the antibodies aswell as pharmaceutically acceptable preparations of the antibodies, acontainer comprising the preparation, and a kit comprising the containerwherein the antibody of the invention is made available for the methodsof use to treat a human subject.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the epitope revealed by X-ray diffraction analysis of aco-crystal of 10H10 Fab or with a human adapted variant (M1593 Fab) andhuman TF-ECD residues 5-208, where the two contact residues that werechanged in M1593 H-CDR1 (T31P) and HCDR-2 (S57F) are shown.

FIG. 2 is an alignment of the amino acid residues of human (SEQ ID NO:1, 1-219), cyno (SEQ ID NO: 2, 1-220), and mouse TF-ECD (SEQ ID NO: 3,1-221) showing residue positions contacted by the murine antibodyTF8-5G9 (Huang et al. 1998 J Mol Biol 275:873-94) and 10H10 and thoseresidues known to be in contact with the coagulation factors FVII/VIIaand FX.

FIG. 3 shows the three dimensional projection of human TF-ECD with theareas indicated contacted by the paratopes of 5G9 and 10H10 as well asthe coagulation factors FVII and FX, where only residues L104 and T197are contacted by both 10H10 and FX.

FIG. 4 shows an alignment of the amino acid sequences of the heavy chain(upper alignment) and light chain (lower alignment) variable domains ofthe murine antibody 10H10 (SEQ ID NO: 4 and 5, respectively), the humanframework adapted sequences of antibody M59 (SEQ ID NO: 19 and 23,respectively) and, two selected affinity matured variable domainsequences H116 (SEQ ID NO: 133) and H171 (SEQ ID NO: 139).

FIG. 5 shows the relative percent inhibition by the 27 affinity maturedmAbs to FVIIa-induced IL-8 release at 0.24 ug/ml by MDB-MB-231 breastcancer cells compared to the isotype control B37.

FIG. 6 shows a plot of tumor volume over days post-implantation ofMDA-MB231 tumor cells in immunocompromised mice where the group dosedwith M1593 reduced growth of an established tumor.

FIG. 7 shows a plot of tumor volume over days post-implantation of A431human squamous tumor cells in immunocompromised mice where the groupdosed with M1593 reduced growth of an established tumor.

FIG. 8 shows a plot of the percent target cell lysis (MDA-MB231 cells)by human PBMC versus MAb concentrations for the murine variabledomain-human IgG1 (M1), murine variable domain-human IgG4 with alaninesubstitution at positions 234 and 235, M1593 as wild-type IgG1 producedin unmodified CHO, as M1593-LF produced in a CHO line selected forproducing glycan with low fucose content, and M1593-DE with Kabatposition substitutions at S239D and 1332E.

DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO: Description Features or Origin 1 Human Tissue Factor ECD =1-219 Mature Chain 2 Cynomolgous Monkey ECD only 1-220 Tissue Factor ECD3 Mus musculus Tissue ECD = 1-221 Factor (P20352) 4 10H10 Heavy Chain(HC) Variable Region 5 10H10 Light Chain (LC) Variable Region 6 H-CDR1of 10H10 7 H-CDR2 of 10H10 8 H-CDR3 of 10H10 9 L-CDR1 of 10H10 10 L-CDR2of 10H10 11 L-CDR3 of 10H10 12 H15 IGHV5-a 13 H16 IGHV1-46 14 H17IGHV1-3 15 H18 IGHV3-74 16 H19 IGHV1-69 17 H20 IGHV1-18 18 H21 IGHV1-f19 H22 s1_IGHV5-a 20 H23 s1_IGHV1-69 21 H24 s1_IGHV1-f 22 L2 IGKV4-1_B323 L3 IGKV2D40_O1 24 L4 IGKV2D-28_A3 25 L5 IGKV2D-29_A2 26 L7IGKV2-24_A23 27 H-CDR2 of H22, H23, Murine, Kabat -7 and H24 28 FR1 ofH15 and H22 IGHV5-a 29 FR1 of H16, H17 and IGHV1-6, IGHV1- H20 3,IGHV1-18 30 FR1 of H18 IGHV3-74 31 FR1 of H19 and H23 IGHV1-69 32 FR1 ofH21 and H24 33 FR2 of H15 and H22 IGHV5-a 34 FR2 of H16, H19, H20, FR2of IGHV1-46, and H23_s1_IGHV1-69 IGHV1-69, IGHV1-18, and s1_IGHV1-69 35FR2 of H17 IGHV1-3 36 FR2 of H18 IGHV3-74 37 FR2 of H21 and H24 IGHV1-f38 FR3 of H15 IGHV5-a 39 FR3 of H16 IGHV1-46 40 FR3 of H17 IGHV1-3 41FR3 of H18 IGHV3-74 42 FR3 of H19 IGHV1-69 43 FR3 of H20 IGHV1-18 44 FR3of H21 IGHV1-f 45 FR3 of H22 s1_IGHV5-a 46 FR3 of H23 s1_IGHV1-69_(—) 47FR3 of H24 s1_IGHV1-f 48 FR1 of L2 IGKV4-1_B3 49 FR1 of L3 IGKV2D40_O150 FR1 of L4 IGKV2D-28_A3 51 FR1 of L5 IGKV2D-29_A2 52 FR1 of L7IGKV2-24_A23 53 FR2 of L2 IGKV4-1_B3 54 FR2 of L3 & L4 IGKV2D-28_A3 55FR2 of L5 IGKV2D-29_A2 56 FR2 of L7 IGKV2-24_A23 57 FR3 of L2 IGKV4-1_B358 FR3 of L3, L4, and L5 IGKV2D40_O1, IGKV2D-28_A3, IGKV2D-29_A2 59 FR3of L7 IGKV2-24_A23 60 FR4 HC IGHJ4 61 FR4 LC IGKJ2 62 H-CDR1 of H106 inM1602 63 H-CDR1 of H116 in M1587 64 H-CDR1 of H117 in M1590 65 H-CDR1 ofH122 in M1591 66 H-CDR1 of H133 in M1612 67 H-CDR1 of H134 in M1597 68H-CDR1 of H136 in M1613, and H185 of M1596 69 H-CDR1 of H136 in M1613 70H-CDR1 of H139 in M1585 71 H-CDR1 of H158 in M1594 72 H-CDR1 of H160 inM1595 M1595 73 H-CDR1 of H164 in M1586 74 H-CDR1 of H165 in M1592 75H-CDR1 of H168 in M1605 76 H-CDR1 of H171 in M1593 77 H-CDR1 of H173 inM1584 78 H-CDR1 of H179 in M1588 79 H-CDR1 of H181 in M1606 80 H-CDR1 ofH187 in M1589 81 H-CDR1 of H189 in M1607 82 H-CDR1 of H177, H130, H105,and H128 83 H-CDR1 variants 84 H-CDR2 of H106 in M1602 85 H-CDR2 of H115in M1610 86 H-CDR2 of H116 in M1587 87 H-CDR2 of H117 in M1590 88 H-CDR2of H128 in M1611 89 H-CDR2 of H130 in M1599 90 H-CDR2 of H134 in M159791 H-CDR2 of H136 in M1613 92 H-CDR2 of H137 in M1598 93 H-CDR2 of H138in M1604 94 H-CDR2 of H160 in M1595 95 H-CDR2 of H164 in M1586 96 H-CDR2of H165 in M1592 97 H-CDR2 of H168 in M1605 98 H-CDR2 of H171 in M159399 H-CDR2 of H173 in M1584 100 H-CDR2 of H177 in M1583 101 H-CDR2 ofH179 in M1588 102 H-CDR2 of H181 in M1606 103 H-CDR2 of H185 in M1596104 H-CDR2 of H187 in M1589 105 H-CDR2 of H189 in M1607 106 H-CDR2 ofH207 in M1608 107 H-CDR2 variants 108 L-CDR1 of L138 in both M1646 &M1638 109 L-CDR1 of L162 in both M1651 & M1643 110 L-CDR1 of L225 inboth M1652 & M1644 111 L-CDR1 of L283 in both M1653 & M1645 112 L-CDR1of L320 in both M1647 & M1639 113 L-CDR1 of L327 in both M1648 & M1640114 L-CDR1 of L335 in both M1649 & M1641 115 L-CDR1 of L369 in bothM1650 & M1642 116 L-CDR1 variants 117 L-CDR2 of L138 in both M1646 &M1638 118 L-CDR2 of L320 in both M1647 & M1639 119 L-CDR2 of L335 inboth M1649 & M1641 120 L-CDR2 Variants 121 L-CDR3 of L162 in both M1651& M1643 122 L-CDR3 of L225 in both M1652 & M1644 123 L-CDR3 of L283 inboth M1653 & M1645 124 L-CDR3 of L320 in both M1647 & M1639 125 L-CDR3of L327 in both M1648 & M1640 126 L-CDR3 of L335 in both M1649 & M1641127 L-CDR3 of L369 in both M1650 & M1642 128 L-CDR3 Variants 129 H177130 H173 131 H139 132 H164 133 H116 134 H179 135 H187 136 H117 137 H122138 H165 139 H171 140 H158 141 H160 142 H185 143 H134 144 H137 145 H130146 H105 147 H106 148 H138 149 H168 150 H181 151 H189 152 H207 153 H115154 H128 155 H133 156 H136 157 L138 158 L320 159 L327 160 L335 161 L369162 L162 163 L225 164 L283 165 M1593 full-length light chain 166 M1593full-length heavy chain 167 M1593-DE full-length heavy chain

DETAILED DESCRIPTION OF THE INVENTION Abbreviations

TF=Tissue Factor, huTF=Human Tissue Factor, muTF=Murine Tissue Factor,cynoTF=Cynomolgus Tissue Factor, TF-FVIIa=Tissue Factor-Factor VIIacomplex, TF/FVIIa=Tissue Factor-Factor VIIa complex, HC=Heavy chain,LC=Light chain, v-region=variable region, VH=Heavy chain variableregion, VL=Light chain variable region, CCD=Charge-coupled device,CDR=Complementarity determining region,CHES=2-(N-cyclohexylamino)-ethanesulfonic acid,EDTA=Ethylenediaminetetraacetic acid, ECD=Extracellular domain,HEPES=N-(2-hydroxyethyl)-piperazine-N′-2-ethanesulfonic acid, HEK=Humanembryonic kidney cells, MES=2-(N-morpholino)ethanesulfonic acid,PAR=Protease activated receptor, PBMC=peripheral blood mononuclearcells, PBS=Phosphate buffered saline, PDB=Protein Data Bank,PEG=Polyethylene glycol, SDS PAGE=Sodium dodecyl sulfate polyacrylamidegel electrophoresis, SEC=Size exclusion chromatography, MAb=Monoclonalantibody, FR=Framework in antibody, HFA=Human Framework adaption.

DEFINITIONS & EXPLANATION OF TERMINOLOGY

As used herein, an “antibody” includes whole antibodies and any antigenbinding fragment or a single chain thereof. Thus, the antibody includesany protein or peptide containing molecule that comprises at least aportion of an immunoglobulin molecule, such as but not limited to, atleast one complementarity determining region (CDR) of a heavy or lightchain or a ligand binding portion thereof, a heavy chain or light chainvariable region, a heavy chain or light chain constant region, aframework (FR) region, or any portion thereof, or at least one portionof a binding protein, which can be incorporated into an antibody of thepresent invention. The term “antibody” is further intended to encompassantibodies, digestion fragments, specified portions and variantsthereof, including antibody mimetics or comprising portions ofantibodies that mimic the structure and/or function of an antibody or aspecified fragment or portion thereof, including single chain and singledomain antibodies and fragments thereof. Functional fragments includeantigen-binding fragments to a preselected target. Examples of bindingfragments encompassed within the term “antigen binding portion” of anantibody include (i) a Fab fragment, a monovalent fragment consisting ofthe VL, VH, CL and CH, domains; (ii) a F(ab′)2 fragment, a bivalentfragment comprising two Fab fragments linked by a disulfide bridge atthe hinge region; (iii) a Fd fragment consisting of the VH and CH,domains; (iv) a Fv fragment consisting of the VL and VH domains of asingle arm of an antibody, (v) a dAb fragment (Ward et al., (1989)Nature 341:544-546), which consists of a VH domain; and (vi) an isolatedcomplementarity determining region (CDR). Furthermore, although the twodomains of the Fv fragment, VL and VH, are coded for by separate genes,they can be joined, using recombinant methods, by a synthetic linkerthat enables them to be made as a single protein chain in which the VLand VH regions pair to form monovalent molecules (known as single chainFv (scFv); see e.g., Bird et al. (1988) Science 242:423-426, and Hustonet al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such singlechain antibodies are also intended to be encompassed within the term“antigen-binding portion” of an antibody. These antibody fragments areobtained using conventional techniques known to those with skill in theart, and the fragments are screened for utility in the same manner asare intact antibodies. Conversely, libraries of scFv constructs can beused to screen for antigen binding capability and then, usingconventional techniques, spliced to other DNA encoding human germlinegene sequences. One example of such a library is the “HuCAL: HumanCombinatorial Antibody Library” (Knappik, A. et al. J Mol Biol (2000)296(1):57-86).

The term “CDR” refers to the complementarity determining region orhypervariable region amino acid residues of an antibody that participatein or are responsible for antigen-binding. The hypervariable region orCDRs of the human IgG subtype of antibody comprise amino acid residuesfrom residues 24-34 (L-CDR1), 50-56 (L-CDR2) and 89-97 (L-CDR3) in thelight chain variable domain and 31-35 (H-CDR1), 50-65 (H-CDR2) and95-102 (H-CDR3) in the heavy chain variable domain as described by Kabatet al. (1991 Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.)and/or those residues from a hypervariable loop (i.e., residues 26-32(L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and26-32 (H1), 52-56 (H2), and 95-101 (H3) in the heavy chain variabledomain as described by (Chothia and Lesk, 1987 J. Mol. Biol. 196:901-917). Chothia and Lesk refer to structurally conserved hypervariableloops as “canonical structures”. Framework or FR1-4 residues are thosevariable domain residues other than and bracketing the hypervariableregions. The numbering system of Chothia and Lesk takes into accountdifferences in the number of residues in a loop by showing the expansionat specified residues denoted by the small letter notations, e.g. 30a,30b, 30c, etc. More recently, a universal numbering system has beendeveloped and widely adopted, international ImMunoGeneTics informationSystem® (IMGT) (LaFranc, et al. 2005. Nucl Acids Res. 33:D593-D597).

Herein, the CDRs are referred to in terms of both the amino acidsequence and the location within the light or heavy chain by sequentialnumbering. As the “location” of the CDRs within the structure of theimmunoglobulin variable domain is conserved between species and presentin structures called loops, by using numbering systems that alignvariable domain sequences according to structural features, CDR andframework residues and are readily identified. This information is usedin grafting and replacement of CDR residues from immunoglobulins of onespecies into an acceptor framework from, typically, a human antibody.

The terms “Fc,” “Fc-containing protein” or “Fc-containing molecule” asused herein refer to a monomeric, dimeric or heterodimeric proteinhaving at least an immunoglobulin CH2 and CH3 domain. The CH2 and CH3domains can form at least a part of the dimeric region of theprotein/molecule (e.g., antibody).

The term “epitope” means a protein determinant capable of specificbinding to an antibody. Epitopes usually consist of chemically activesurface groupings of molecules such as amino acids or sugar side chainsand usually have specific three-dimensional structural characteristics,as well as specific charge characteristics. Conformational andnonconformational epitopes are distinguished in that the binding to theformer but not the latter is lost in the presence of denaturingsolvents.

As used herein, K_(D) refers to the dissociation constant, specifically,the antibody K_(D) for a predetermined antigen, and is a measure ofaffinity of the antibody for a specific target. High affinity antibodieshave a K_(D) of 10⁻⁸ M or less, more preferably 10⁻⁹ M or less and evenmore preferably 10⁻¹⁰ M or less, for a predetermined antigen. Thereciprocal of K_(D) is K_(A), the association constant. The term“k_(dis)” or “k₂,” or “k_(d)” as used herein, is intended to refer tothe dissociation rate of a particular antibody-antigen interaction. The“K_(D)” is the ratio of the rate of dissociation (k₂), also called the“off-rate (k_(off))” to the rate of association rate (k₁) or “on-rate(k_(on)).” Thus, K_(D) equals k₂/k₁ or k_(off)%_(on) and is expressed asa molar concentration (M). It follows that the smaller the K_(D), thestronger the binding. Thus, a K_(D) of 10⁻⁶ M (or 1 microM) indicatesweak binding compared to 10⁻⁹ M (or 1 nM).

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A monoclonal antibody composition displays asingle binding specificity and affinity for a particular epitope. Theterm also includes “recombinant antibody” and “recombinant monoclonalantibody” as all antibodies are prepared, expressed, created or isolatedby recombinant means, such as (a) antibodies isolated from an animal ora hybridoma prepared by the fusion of antibody secreting animal cellsand an fusion partner, (b) antibodies isolated from a host celltransformed to express the antibody, e.g., from a transfectoma, (c)antibodies isolated from a recombinant, combinatorial human or otherspecies antibody library, and (d) antibodies prepared, expressed,created or isolated by any other means that involve splicing ofimmunoglobulin gene sequences to other DNA sequences. An “isolatedantibody,” as used herein, is intended to refer to an antibody which issubstantially free of other antibodies having different antigenicspecificities. An isolated antibody that specifically binds to anepitope, isoform or variant of human TF may, however, havecross-reactivity to other related antigens, e.g., from other species(e.g., TF species homologs). Moreover, an isolated antibody may besubstantially free of other cellular material and/or chemicals. In oneembodiment of the invention, a combination of “isolated” monoclonalantibodies having different specificities are combined in a well definedcomposition.

As used herein, “specific binding,” “immunospecific binding” and “bindsimmunospecifically” refers to antibody binding to a predeterminedantigen. Typically, the antibody binds with a dissociation constant(K_(D)) of 10⁻⁷ M or less, and binds to the predetermined antigen with aK_(D) that is at least twofold less than its K_(D) for binding to anon-specific antigen (e.g., BSA, casein, or any other specifiedpolypeptide) other than the predetermined antigen. The phrases “anantibody recognizing an antigen” and “an antibody specific for anantigen” are used interchangeably herein with the term “an antibodywhich binds specifically to an antigen.” As used herein “highlyspecific” binding means that the relative K_(D) of the antibody for thespecific target epitope is at least 10-fold less than the K_(D) forbinding that antibody to other ligands.

As used herein, “isotype” refers to the antibody class (e.g., IgM orIgG) that is encoded by heavy chain constant region genes. Some antibodyclasses further encompass subclasses which are also encoded by the heavychain constant regions and further decorated by oligosaccharides atspecific residues within the constant region domains (e.g. IgG1, IgG2,IgG3 and IgG4) which further impart biological functions to theantibody. For example, in human antibody isotypes IgG1, IgG3 and to alesser extent, IgG2 display effector functions as do murine IgG2aantibodies.

By “effector” functions or “effector positive” is meant that theantibody comprises domains distinct from the antigen specific bindingdomains capable of interacting with receptors or other blood componentssuch as complement, leading to, for example, the recruitment ofmacrophages and events leading to destruction of cells bound by theantigen binding domains of the antibody. Antibodies have severaleffector functions mediated by binding of effector molecules. Forexample, binding of the C1 component of complement to antibodiesactivates the complement system. Activation of complement is importantin the opsonisation and lysis of cell pathogens. The activation ofcomplement stimulates the inflammatory response and may also be involvedin autoimmune hypersensitivity. Further, antibodies bind to cells viathe Fc region, with a Fc receptor site on the antibody Fc region bindingto a Fc receptor (FcR) on a cell. There are a number of Fc receptorswhich are specific for different classes of antibody, including IgG(gamma receptors), IgE (eta receptors), IgA (alpha receptors) and IgM(mu receptors). Binding of antibody to Fc receptors on cell surfacestriggers a number of important and diverse biological responsesincluding engulfment and destruction of antibody-coated particles,clearance of immune complexes, lysis of antibody-coated target cells bykiller cells (called antibody-dependent cell-mediated cytotoxicity, orADCC), release of inflammatory mediators, placental transfer and controlof immunoglobulin production.

The terms “tissue factor protein”, “tissue factor” and “TF” are used torefer to a polypeptide having an amino acid sequence corresponding to anaturally occurring human tissue factor or a recombinant tissue factoras described below. Naturally occurring TF includes human species aswell as other animal species such as rabbit, rat, porcine, non humanprimate, equine, murine, and ovine tissue factor (see, for example,Hartzell et al., (1989) Mol. Cell. Biol., 9:2567-2573; Andrews et al.,(1991) Gene, 98:265-269; and Takayenik et al., (1991) Biochem. Biophys.Res. Comm., 181:1145-1150). The amino acid sequence of human tissuefactor is given by the UniProt record P13726 (SEQ ID NO: 1), cynomolgousmonkey (SEQ ID NO: 2), and murine by UniProt P20352 (SEQ ID NO: 3). Theamino acid sequence of the other mammalian tissue factor proteins aregenerally known or obtainable through conventional techniques.

The antibodies of the invention are useful for administering to a humansubject or for contacting human tissue where it is desired to block thefunctions of human TF expressed on a cell, tissue, or organ resultingfrom TF signaling and wherein it is also desired to not substantiallyalter the procoagulant functions of TF resulting from the formation of aTF:FVIIa complex. Such uses can be found in the treatment of tumors, inparticular, primary or secondary solid tumors of the breast, prostate,lung, pancreas, and ovary.

The invention also encompasses nucleic acids encoding the antibodysequences of the invention which can be combined with those sequencesknown in the art to be useful in the construction and manufacturethrough recombinant means or transfer of the information for expressionof the antibodies in a milieu where it is desired that they be formed,such in culture, in situ, and in vivo. The means for the operation ofsuch nucleic acids with the intent of producing an antibody of theinvention are well known to those skilled in the art.

The invention further provides for preparations such as pharmaceuticallyacceptable or stable preparations for administration and storage of anantibody of the invention in isolated form.

1. Composition of the Antibody Properties

The present invention is based on the unexpected discovery that anon-coagulation blocking murine antibody which binds to human TF, knownas 10H10 (Edgington, et al. U.S. Pat. No. 5,223,427) is capable ofabrogating the signaling of TF in certain cells (Ahmed, et al. 2006,cited above, WO2007/056352A2). Therefore, an antibody of the inventionis one that which retains the binding epitope of the murine antibody10H10, which antibody does not compete with tissue factor for FVIIabinding and does not substantially block the procoagulant, amidolyticactivity of the TF-VIIa complex and which does block TF-VIIa mediatedsignaling and downstream oncogenic effects such as cytokine IL-8release. The antibody of the invention is adapted to human germline IgGgenes as represented in the IMGT database and retains binding to humanTF while not interfering with the ability of TF to initiate coagulationin the presence of calcium in human plasma.

An antibody that retains the binding epitope of murine antibody 10H10can be assessed generally by assessing the ability of the antibody bindto TF and to compete with 10H10 for binding to human TF while at thesame time, when present in a sample comprising TF in the presence ofhuman plasma, will not substantially prolong the time required for theTF initiated coagulation of the plasma as compared to a like sample ofhuman plasma in the absence of the antibody. In another sense, theepitope of the antibody can be physically mapped using techniques knownin the art, including but not limited to deletion mutagenesis,substitution mutagenesis, limited proteolysis of TF bound by theantibody followed by peptide fragment identification, andco-crystallization and X-ray diffraction methods to map proximity ofatomic structures of the primary structures of TF and the antibodybinding domains thereby defining a three-dimensional association betweenthe antibody and human TF (FIG. 1).

The epitope, thus, can be defined as non-overlapping with the FVIIabinding site (FIGS. 2 and 3). More specifically, the epitope bound bythe antibody of the invention may contact one or more residues in theN-domain of TF (residues 1-104 of the mature chain as represented by SEQID NO: 1) not contacted by FVII, such as residues 65-70, and not contactresidues K165 and K166 in the C-domain, which are important forsubstrate binding (Kirchofer et al. 2000 Thromb Haemostat 84: 1072-81)while not interfering with the ability of TF to initiate coagulation inthe presence of calcium in human plasma.

In one embodiment the on-rate (k_(a) in 1/M·s) of the antibody isgreater than 1×10⁻⁵. In another embodiment, the off-rate (k_(d) in 1/s)of the antibody for TF is less than 1.0×10⁻⁵ and the resulting K_(D) isless than 1×10⁻⁹ M (less than 1 nM). In a particular embodiment, theantibody is a human germline gene adapted antibody with a K_(D) lessthan 0.5×10⁻⁹ M. In one embodiment the antibody has binding domainsselected from those of the heavy and light chain pairings as shown inTable 11 such as M1639, M1645, M1647, M1652, M1641, M1644, M1587, M1604,M1593, M1606, M1584, M1611, M1596, M1601, M1588, M1594, M1607, M1612,M1595, M1599, M1589, M1592, M1583, and M1610.

The antibody composition may be further characterized as comprising asequence of amino acid residues in the binding domain selected from oneor more of the amino acid sequences given by SEQ ID NO: 6-166.

Antibody Variants with Altered Fc Functions

As the use of therapeutic monoclonal antibodies produced by recombinantmethods expands, features and properties of these complex compositionsare being explored. While the immunospecific and antigen targetingfeatures generally reside in the variable domains and subdomains such asthe loop ends of the hypervariable regions also known as the CDRs, thecomplex interacts with other receptors and serum components afforded bythe structures formed by the constant domains, such as the Fc portion ofan IgG.

Antibodies and other Fc-containing proteins can be compared forfunctionality by several well-known in vitro assays. In particular,affinity for members of the FcγRI, FcγRII, and FcγRIII family of Fcγreceptors is of interest. These measurements could be made usingrecombinant soluble forms of the receptors or cell-associated forms ofthe receptors. In addition, affinity for FcRn, the receptor responsiblefor the prolonged circulating half-life of IgGs, can be measured, forexample, by BIAcore using recombinant soluble FcRn. Cell-basedfunctional assays, such as ADCC assays and CDC assays, provide insightsinto the likely functional consequences of particular variantstructures. In one embodiment, the ADCC assay is configured to have NKcells be the primary effector cell, thereby reflecting the functionaleffects on the FcγRIIIA receptor. Phagocytosis assays may also be usedto compare immune effector functions of different variants, as canassays that measure cellular responses, such as superoxide orinflammatory mediator release. In vivo models can be used as well, as,for example, in the case of using variants of anti-CD3 antibodies tomeasure T cell activation in mice, an activity that is dependent on Fcdomains engaging specific ligands, such as Fcγ receptors.

2. Generation of Tissue Factor Signal-Blocking Antibodies

An antibody having the features and biologic activity of an antibodydescribed in this application can include or be derived from any mammal,such as, but not limited to, a human, a mouse, a rabbit, a rat, arodent, a primate, a goat, or any combination thereof and includesisolated human, primate, rodent, mammalian, chimeric, human- orprimate-adapted antibodies, immunoglobulins, cleavage products and otherspecified portions and variants thereof. Monoclonal antibodies may beprepared by any method known in the art such as the hybridoma technique(Kohler and Milstein, 1975, Nature, 256:495-497) and related methodsusing immortalized fusion partners fused to B-cells. Antibodies for usein the invention may also be generated using single lymphocyte antibodymethods by cloning and expressing immunoglobulin variable region cDNAsgenerated from single lymphocytes selected for the production ofspecific antibodies by for example the methods described by Babcook, J.et al., 1996, Proc. Natl. Acad. Sci. USA 93(15):7843-78481; WO92/02551;WO2004/051268 and International Patent Application number WO2004/106377.

The antibodies, including the target binding domains or subdomains, theconstant domains, and the functional non-target binding domains such asthe Fc-domain as described herein can be derived in several ways wellknown in the art. In one aspect, the sequences of naturally occurringantibody domains are conveniently obtained from published or on-linedocuments or databases, such as V-base (provided by the MRC Centre forProtein Engineering), the National Center for Biologics Information(NCBI Ig blast), or the ImMunoGeneTics (IMGT) database provided by theInternational Immunogenetics Information System®.

Human Antibodies

The invention further provides human immunoglobulins (or antibodies)which bind human TF. These antibodies can also be characterized asengineered or adapted. The immunoglobulins have variable region(s)substantially from a human germline immunoglobulin and include directedvariations in residues known to participate in antigen recognition, e.g.the CDRs of Kabat or the hypervariable loops as structurally defined.The constant region(s), if present, are also substantially from a humanimmunoglobulin. The human antibodies exhibit K_(D) for TF of at leastabout 10⁻⁶ M (1 microM), about 10⁻⁷ M (100 nM), 10⁻⁹ M (1 nM), or less.To affect a change in affinity, e.g., improve affinity or reduce K_(D),of the human antibody for TF, substitutions in either the CDR residuesor other residues may be made.

The source for production of human antibody which binds to TF ispreferably the sequences provide herein as the variable regionscomprising a sequence selected from SEQ ID NO: 129-163, a FR selectedfrom SEQ ID NO: 28-61, and CDRs, where the CDRs are selected from one ormore of SEQ ID NO: 6-11, 27, 62-128 identified as capable of bindinghuman TF and cross-reacting with cynomolgous monkey TF using arepertoire of human derived Fab displayed on filamentous phageparticles.

The substitution of any of non-human CDRs into any human variable domainFR may not allow the same spatial orientation provided by theconformation to the parent variable FR from which the CDRs originated.The heavy and light chain variable framework regions to be paired in thefinal MAb can be derived from the same or different human antibodysequences. The human antibody sequences can be the sequences ofnaturally occurring human antibodies, be derived from human germlineimmunoglobulin sequences, or can be consensus sequences of several humanantibody and/or germline sequences.

Suitable human antibody sequences are identified by computer comparisonsof the amino acid sequences of the mouse variable regions with thesequences of known human antibodies. The comparison is performedseparately for heavy and light chains but the principles are similar foreach.

With regard to the empirical method, it has been found to beparticularly convenient to create a library of variant sequences thatcan be screened for the desired activity, binding affinity orspecificity. One format for creation of such a library of variants is aphage display vector. Alternatively, variants can be generated usingother methods for variegation of a nucleic acid sequence encoding thetargeted residues within the variable domain.

Another method of determining whether further substitutions arerequired, and the selection of amino acid residues for substitution, canbe accomplished using computer modeling. Computer hardware and softwarefor producing three-dimensional images of immunoglobulin molecules arewidely available. In general, molecular models are produced startingfrom solved structures for immunoglobulin chains or domains thereof. Thechains to be modeled are compared for amino acid sequence similaritywith chains or domains of solved three dimensional structures, and thechains or domains showing the greatest sequence similarity is/areselected as starting points for construction of the molecular model. Thesolved starting structures are modified to allow for differences betweenthe actual amino acids in the immunoglobulin chains or domains beingmodeled, and those in the starting structure. The modified structuresare then assembled into a composite immunoglobulin. Finally, the modelis refined by energy minimization and by verifying that all atoms arewithin appropriate distances from one another and that bond lengths andangles are within chemically acceptable limits.

Because of the degeneracy of the code, a variety of nucleic acidsequences will encode each immunoglobulin amino acid sequence. Thedesired nucleic acid sequences can be produced by de novo solid-phaseDNA synthesis or by PCR mutagenesis of an earlier prepared variant ofthe desired polynucleotide. All nucleic acids encoding the antibodiesdescribed in this application are expressly included in the invention.

The variable segments of human antibodies produced as described hereinare typically linked to at least a portion of a human immunoglobulinconstant region. The antibody will contain both light chain and heavychain constant regions. The heavy chain constant region usually includesCH1, hinge, CH2, CH3, and, sometimes, CH4 domains.

The human antibodies may comprise any type of constant domains from anyclass of antibody, including IgM, IgG, IgD, IgA and IgE, and anysubclass (isotype), including IgG1, IgG2, IgG3 and IgG4. When it isdesired that the humanized antibody exhibit cytotoxic activity, theconstant domain is usually a complement-fixing constant domain and theclass is typically IgG₁. When such cytotoxic activity is not desirable,the constant domain may be of the IgG₂ class. The humanized antibody maycomprise sequences from more than one class or isotype.

Nucleic acids encoding humanized light and heavy chain variable regions,optionally linked to constant regions, are inserted into expressionvectors. The light and heavy chains can be cloned in the same ordifferent expression vectors. The DNA segments encoding immunoglobulinchains are operably linked to control sequences in the expressionvector(s) that ensure the expression of immunoglobulin polypeptides.Such control sequences include a signal sequence, a promoter, anenhancer, and a transcription termination sequence (see Queen et al.,Proc. Natl. Acad. Sci. USA 86, 10029 (1989); WO 90/07861; Co et al., J.Immunol. 148, 1149 (1992), which are incorporated herein by reference intheir entirety for all purposes).

The antibodies or Fc or components and domains thereof may also beobtained from selecting from libraries of such domains or components,e.g., a phage library. A phage library can be created by inserting alibrary of random oligonucleotides or a library of polynucleotidescontaining sequences of interest, such as from the B-cells of animmunized animal or human (Hoogenboom, et al. 2000, Immunol. Today 21(8)371-8). Antibody phage libraries contain heavy (H) and light (L) chainvariable region pairs in one phage allowing the expression ofsingle-chain Fv fragments or Fab fragments (Hoogenboom, et al. 2000supra). The diversity of a phagemid library can be manipulated toincrease and/or alter the immunospecificities of the monoclonalantibodies of the library to produce and subsequently identifyadditional, desirable, human monoclonal antibodies. For example, theheavy (H) chain and light (L) chain immunoglobulin molecule encodinggenes can be randomly mixed (shuffled) to create new HL pairs in anassembled immunoglobulin molecule. Additionally, either or both the Hand L chain encoding genes can be mutagenized in a complementaritydetermining region (CDR) of the variable region of the immunoglobulinpolypeptide, and subsequently screened for desirable affinity andneutralization capabilities. Antibody libraries also can be createdsynthetically by selecting one or more human FR sequences andintroducing collections of CDR cassettes derived from human antibodyrepertoires or through designed variation (Kretzschmar and von Ruden2000, Current Opinion in Biotechnology, 13:598-602). The positions ofdiversity are not limited to CDRs, but can also include the FR segmentsof the variable regions or may include other than antibody variableregions, such as peptides.

Other libraries of target binding or non-target binding components whichmay include other than antibody variable regions are ribosome display,yeast display, and bacterial displays. Ribosome display is a method oftranslating mRNAs into their cognate proteins while keeping the proteinattached to the RNA. The nucleic acid coding sequence is recovered byRT-PCR (Mattheakis, L. C. et al. 1994. Proc. Natl. Acad. Sci. USA 91,9022). Yeast display is based on the construction of fusion proteins ofthe membrane-associated alpha-agglutinin yeast adhesion receptor, aga1and aga2, a part of the mating type system (Broder, et al. 1997. NatureBiotechnology, 15:553-7). Bacterial display is based on fusion of thetarget to exported bacterial proteins that associate with the cellmembrane or cell wall (Chen and Georgiou 2002. Biotechnol Bioeng,79:496-503).

The invention also provides for nucleic acids encoding the compositionsof the invention as isolated polynucleotides or as portions ofexpression vectors including vectors compatible with prokaryotic,eukaryotic or filamentous phage expression, secretion and/or display ofthe compositions or directed mutagens thereof

3. Methods of Producing the Antibody of the Invention

Once an antibody molecule of the invention has been identified accordingto the structural and functional characteristics described herein,nucleic acid sequences encoding the desired portions of, or the entireantibody chains, can be cloned, copied, or chemically synthesized andcan be isolated and used to express the antibody by routine methods. Theantibody of the invention may be purified by any method known in the artfor purification of an immunoglobulin molecule, for example, bychromatography (e.g., ion exchange, affinity, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins. In addition,the antibodies of the present invention or fragments thereof can befused to heterologous polypeptide sequences described herein orotherwise known in the art, to facilitate purification.

Host Cell Selection or Host Cell Engineering

As described herein, the host cell chosen for expression of therecombinant Fc-containing protein or monoclonal antibody is an importantcontributor to the final composition, including, without limitation, thevariation in composition of the oligosaccharide moieties decorating theprotein in the immunoglobulin CH2 domain. Thus, one aspect of theinvention involves the selection of appropriate host cells for useand/or development of a production cell expressing the desiredtherapeutic protein.

Further, the host cell may be of mammalian origin or may be selectedfrom COS-1, COS-7, HEK293, BHK21, CHO, BSC-1, Hep G2, 653, SP2/0, 293,HeLa, myeloma, lymphoma, yeast, insect or plant cells, or anyderivative, immortalized or transformed cell thereof.

Alternatively, the host cell may be selected from a species or organismincapable of glycosylating polypeptides, e.g. a prokaryotic cell ororganism, such as and of the natural or engineered E. coli spp,Klebsiella spp., or Pseudomonas spp.

4. Methods of Using an Anti-TF Antibody

The compositions (antibody, antibody variants, or fragments) generatedby any of the above described methods may be used to diagnose, treat,detect, or modulate human disease or specific pathologies in cells,tissues, organs, fluid, or, generally, a host. As taught herein,modification of the Fc portion of an antibody, Fc-fusion protein, or Fcfragment to provide a more specifically suited range of effectorfunctions after target binding but where in the antibody retains theoriginal targeting properties will generate variants of the antibody forspecific applications and therapeutic indications.

The diseases or pathologies that may be amenable to treatment using acomposition provided by the invention include, but are not limited to:cancer; including primary solid tumors and metastases; carcinomas,adenocarcinomas, melanomas, liquid tumors such as lymphomas, leukemiasand myelomas and invasive masses formed as the cancer progresses; softtissue cancers; sarcomas, osteosarcoma, thymoma, lymphosarcoma,fibrosarcoma, leiomyosarcoma, lipomas, glioblastoma, astrosarcoma,cancer of the prostate, breast, ovary, stomach, pancreas, larynx,esophagus, testes, liver, parotid, biliary tract, colon, rectum, cervix,uterus, endometrium, thyroid, lung, kidney, or bladder.

In so far as the antibody of the invention reduces the pro-oncogenicmilieu in a tissue by blocking the ability of TF to participate in thedownstream release of cytokines such as the inflammatory cytokine, IL-8,the antibody of the invention can be used prophylactically or inconjunction with other treatments directed to suppressing tumorproliferation and angiogenesis. Most age-related cancers derive from theepithelial cells of renewable tissues. An important element ofepithelial tissues is the stroma, the sub-epithelial layer composed ofextracellular matrix and several cell types including fibroblasts,macrophages, and endothelial cells. In cancerous tumors the stroma iscritical for tumor growth and progression and TF can be expressed onstromal cells as well as the cancerous epithelial cells. Therefore, thepresence of the downstream factors resulting from TF:VIIa signaling instroma may create a pro-oncogenic tissue environment that synergizeswith oncogenic mutations to drive the formation of neoplastic tissue.

Similarly, when TF is expressed in adipose tissue it can modify thefunction of the tissue in conditions such as obesity, metabolicsyndrome, and diabetes. The antibody of the invention can be useful intreating these conditions by blocking TF:VIIa signaling. Some of thefactors produced downstream of TF:FVIIa signaling, including IL-8 andIL-6, are powerful mediators of inflammation. Additional uses of theantibody of the invention therefore include treatment of inflammatoryconditions such as, but not limited to, rheumatoid arthritis,inflammatory bowel disease, and asthma.

As the antibody of the invention inhibits TF:VIIa signaling and reducesdownstream effects promoting angiogenesis, the antibodies of theinvention can be useful in treating other diseases, disorders, and/orconditions, in addition to cancers, which involve angiogenesis. Thesediseases, disorders, and/or conditions include, but are not limited to:benign tumors, for example hemangiomas, acoustic neuromas,neurofibromas, trachomas, and pyogenic granulomas; artherosclericplaques; ocular angiogenic diseases, for example, diabetic retinopathy,retinopathy of prematurity, macular degeneration, corneal graftrejection, neovascular glaucoma, retrolental fibroplasia, rubeosis,retinoblastoma, uveitis and Pterygia (abnormal blood vessel growth) ofthe eye; rheumatoid arthritis; psoriasis; delayed wound healing;endometriosis; vasculogenesis; granulations; hypertrophic scars(keloids); nonunion fractures; scleroderma; trachoma; vascularadhesions; myocardial angiogenesis; coronary collaterals; cerebralcollaterals; arteriovenous malformations; ischemic limb angiogenesis;Osler-Webber Syndrome; plaque neovascularization; telangiectasia;hemophiliac joints; angiofibroma; fibromuscular dysplasia; woundgranulation; Crohn's disease; and atherosclerosis.

In so far as the antibody of the invention inhibits TF:VIIa signaling,the antibody can be used to treat and/or diagnose hyperproliferativediseases, disorders, and/or conditions, including but not limited toneoplasms. The antibody can inhibit proliferation of the disorderthrough direct or indirect interactions. Examples of hyperproliferativediseases, disorders, and/or conditions that can be treated, and/ordiagnosed by the antibodies of the invention, include,hyperproliferative diseases, disorders, and/or conditions include, butare not limited to, hypergammaglobulinemia, lymphoproliferativediseases, disorders such as Castleman's disease, and/or conditions,paraproteinemias, purpura, sarcoidosis, Sezary Syndrome, Waldenstron'sMacroglobulinemia, Gaucher's Disease, histiocytosis, and any otherhyperproliferative disease in an organ, tissue or fluid bodycompartment.

Additional ways in which the antibodies of the present invention can beused therapeutically include, but are not limited to, directedcytotoxicity of the antibody, e.g., as mediated by complement (CDC) orby effector cells (ADCC), or indirect cytotoxicity of the antibody,e.g., as immunoconjugates.

While having described the invention in general terms, the embodimentsof the invention will be further disclosed in the following examplesthat should not be construed as limiting the scope of the claims. In theexperimental descriptions, certain reagents and procedures were used toproduce proteins or an antibody or a specified fragment. Analyticalmethods routinely used to characterize the antibody are described below.

Materials and Methods Protein and Antibody Standards

The recombinant extracellular domain (ECD) of human TF was constructedin two forms: for ELISA and Biacore-based direct binding assays, aminoacid 1-219 of the mature chain of TF (SEQ ID NO: 1) was expressed in amammalian system with a C-terminal His6-tag peptide; forco-crystallography studies, amino acid 5-213 of SEQ ID NO: 1 wasexpressed with a C-terminal His6-tag peptide in a bacterial system.Human_(TF1-219) was biotinylated using NHS-ester chemistry targetingamine residues on the protein. For coagulation assays, Innovin® (DadeBehring Inc. cat #B4212), a lyophilized recombinant human tissue factorcombined with phospholipids, calcium, buffers and stabilizers fordiagnostic use was used.

Cynomolgous monkey (cyno) TF-ECD (SEQ ID NO: 2) was cloned using PCRfrom cDNA isolated form cynomolgous testes tissue obtained from BioChainInstitute (Hayward, Calif.).

Several antibodies were used as reference antibodies: i) 10H10 clonedfrom the original hybridoma TF9.10H10-3.2.2 (U.S. Pat. No. 7,223,427)ii) a 10H10 mouse-human chimera, comprising SEQ ID NO: 4 and SEQ ID NO:5 with human IgG1/Kappa constant regions, designated M1 iii) M59, ahuman FR adapted antibody comprising the six CDRs of 10H10 and servingas the parent antibody for affinity maturation, comprising SEQ ID NO:19and SEQ ID NO:23 with human IgG1 and human Kappa constant region; iv)murine anti-human tissue factor antibody TF8-5G9 (U.S. Pat. No.7,223,427); v) a humanized from of the antibody 5G9, known as CNTO 860,as a human IgG1/Kappa (U.S. Pat. No. 7,605,235); and vi) an isotypecontrol (human IgG1/kappa) antibody binding to an irrelevant antigen(RSV) called B37.

Antibody Expression and Purification

Routine procedures were used to express and purify the disclosedantibodies. For primary screening, DNA encoding these molecules weretransiently expressed in 96-well plates in HEK 293E cells and thesupernatants were tested for activity (binding) 96 hours followingtransfection. Hits were identified and chosen for pilot-scale expressionand purification. Pilot-scale expression was done transiently in HEK293F cells or CHO-S at a volume of 750 ml. The harvested supernatantswere purified via Protein A chromatography and the purified proteinswere assessed for their affinity and functional activity. In addition,the purified proteins were subjected to biophysical characterization bySDS-PAGE, SE-HPLC, and cross-interaction chromatography (CIC).Theoretical isoelectric points (pI) were calculated for each variant aswell. From the pilot scale characterizations, a set of final leadcandidates was transfected in WAVE bioreactors and purified via ProteinA chromatography.

Fab Production and Monoclonal Fab ELISA

Glycerol stocks from phage panning rounds were miniprepped and the pIXgene was excised by NheI/SpeI digestion. After religation, the DNA wastransformed into TG-1 cells and grown on LB/Agar plates overnight. Thenext day, colonies were picked, grown overnight, and the cultures usedfor (i) colony PCR and sequencing of the V-regions, and (ii) inductionof Fab production. For Fab production, the overnight culture was diluted10-100 fold in new media and grown for 5-6 hours at 37 degrees C. Fabproduction was induced by the addition of fresh media containing IPTGand the cultures were grown overnight at 30 degrees C. The followingday, the cultures were spun down and the supernatants, containing thesoluble Fab proteins, were used for Fab ELISA.

For Fab ELISA, Fabs were captured onto plates by a polyclonalanti-Fd(CH1) antibody. After appropriate washing and blocking,biotinylated hTF was added at 0.2 nM concentration. This concentrationenables ranking of the Fab variants, defined as percent binding of theparent, in which the parent Fab, present as a control in all plates, isdefined as 100% binding. The biotinylated hTF was detected byHRP-conjugated streptavidin and chemiluminescence read in a platereader.

TF-ECD Binding Mab-Based ELISA

A solution Phase direct TF binding ELISA using chemiluminescentdetection was used to rank the top binders from human frameworkadaptation library. The 96 well black maxisorp plates were coated with100 uL of 4 ug/ml Goat anti human IgG FC diluted incarbonate-bicarbonate buffer, pH 9.4 at 4° C. overnight and then washedthrice with wash buffer (PBS with 0.05% Tween-20 solution) and blockedwith 300 μl 1% BSA/10 mM PBS solution for 1 hour followed by a washingas before. Samples or standards were diluted to 50 ng/ml in Assay Buffer(1% BSA in PBS+05% Tween) and 100 ul was added to the assay plate atroom temperature for 1 hour with shaking. The plates were washed thriceand 100 ul per well of human or cynomologus TF-ECD with His Tag wasadded at 100 ng/ml diluted in Assay Buffer and incubate for 2 hours atroom temperature. After washing, 100 ul per well of Qiagen peroxidaseconjugated penta-his at 1:2000 dilution in assay buffer was added andincubated 1 hour at room temperature with shaking. The BM ChemiLumSubstrate (BM Chemilum, POD, Roche) was made freshly at 1:100 dilutioninto Buffer and 100 ul added to the plates after a final wash. After 10minutes the plates are read on Perkin Elmer Envision Reader, BM ChemiLumprogram.

MDA-MB-231 Whole Cell Binding of Anti-Tissue Factor mAbs by FACS

This assay is used to detect the direct binding of antibody toendogenouse human TF expressed in breast cancer cells. Prepare fourpoint titrations of the test mAbs in FACS buffer (1% FBS in PBS) induplicate. Start a titration at 1,000 ng/ml with 1:4 dilutions. M1, theparent molecule, is used as positive control while B37, anti-RSV mAb, isused as negative/isotype control. Unstained cells and secondaryantibody, Cy-5 conjugated Goat anti-Human IgG Fc antibody in FACS buffer1:200, is used as controls and prepared immediately before use.

Using standard tissue culture technique, rinse adherent MDA-MB-231 cellsin culture flask with once with PBS (w/o Ca+2/Mg+2). Lift cells withVersene and count the cells and seed 200,000 cells per well in apolystyrene V-bottom plate. FACS Analysis Protocol: Tissue FactorBinding. Pellet the cells in a Allegra X-15R centrifuge at 450×g for 3minutes at 4° C., resuspend in FACS Buffer (2% FBS in PBS) and plate200,000 cells per well in 200 uL. Pellet cells at 450×g for 3 minutes at4° C. Discard supernatants and add 100 uL/well test or control mAbs todesignated wells and incubate on ice or at 4° C. for 1 hour (+/−10minutes). Pellet cells at 450×g for 3 minutes at 4° C. Discardsupernatants and wash cells once in FACS buffer. Resuspend cells in 200uL/well FACS buffer and pellet cells at 450×g for 3 minutes at 4° C.Discard supernatants and add 100 uL/well secondary antibody todesignated wells (triterate), and incubate on ice for 1 hour (+/−10minutes). Pellet cells at 450×g for 3 minutes at 4° C. Discardsupernatants and wash cells 2 times in FACS buffer before resuspendingthe cells in 200 uL/well FACS Buffer (triterate). Pellet cells at 450×gfor 3 minutes at 4° C. Discard supernatant and resuspend cells in 100uL/well CytoFix Buffer. Analyze reactions by flow cytometry (BDFACSArray). The FlowJo Software is used for FACS data analysis by gatingthe main population of cells in the unstained control well and applyingthe gate to the whole data set. The data is exported as a table of thegeometric mean fluorescence intensity (MFI) in the red channel for theapplied gate.

Thermafluor Assay

Thermofluor technology is a kinetic measurement of the unfolding of amolecule as it is heated. As the molecule is heated, a dye (ANS) is ableto bind to the molecule as it unfolds. The dye will fluoresce as itbinds to the molecule, and this fluorescence is measured over time. Inthis assay, the unfolding of the antibodies was measured from 37-95° C.,and detected every 0.5° C. The Tms of the parent molecules in bothmurine and chimera form (10H10, M1, 5G9 and CNTO860) were also measured,along with 2 mAbs with known Tms to be used as assay controls (Emmp 4A5,and Emmp 5F6).

This assay was used to predict thermal stability of the human frameworkadaptation library variants. Dilute purified antibodies to 0.5 mg/mL inPBS and add 2 ul sample to each well to total 1 ug sample per well. Eachsample is added in duplicate. Stock ANS is at 500 mM in DMSO. Dilutestock ANS 1:12 into DMSO (to 40 mM); Make Dye/Tween solution bycombining 20 ul of the 40 mM ANS solution, 2.8 ul 10% Tween and 1.98 mLPBS; add 2 uL Dye/Tween solution and 2 ul oil. Centrifuge plates (2 minat 450 rpm). Thermofluor Settings Shutter set to manual, RampTemperature 0.5 C/sec., Continuous Ramp, Temperature Ramp: 50-95° C.Select hold 15 s at high T., Exposure time 10 s/1 rep., Gain normal=2,Select “Single SC Image/plate”.

Cross Interaction Chromatography (CID)

To determine the interaction of the various antibodies with other humanantibodies, chromatography experiments were performed using a columncoupled with human IgG (Sigma Aldrich). Briefly, 50 mgs of human IgGwere coupled to a 1 ml NHS-Sepharose column (GE Healthcare) followingthe manufacturer's instructions. Uncoupled IgG was removed by washingwith 0.1M Tris, pH8, 0.5M NaCl and unreacted NHS groups were blockedwith the same buffer. The coupling efficiency was determined bymeasuring the protein concentration remaining in the unreacted couplingbuffer and washes using Pierce's Coomassie Plus Assay Kit (ThermoPierce) and subtracting from the amount of protein beforeimmobilization. A control column was also prepared using the sameprotocol only no protein was added to the resin.

The control column was run first on a Dionex UltiMate 3000 HPLC afterbeing equilibrated with PBS, pH7 at a flow rate of 0.1 ml/min. 201 ofthe stock protein solution was injected first to ensure non-specificbinding sites were blocked followed by 201 of 10% acetone to check theintegrity of the column.

Samples to be analyzed were diluted to 0.1 mg/ml in PBS, pH7. 20 microLof each sample was injected onto each column and allowed to run at 0.1ml/min for 30 min. Retention times were recorded and the retentionfactor (k′) was calculated for each variant.

The calculation of k′ is the difference in the retention time on aprotein derivatized column (IgG coupled column), t_(R), and theretention time on a column with no protein coupled to it, to. Thecalculation also takes into account the retention time of acetone onboth columns to standardize the column. Acceptable values for k′ areless than 0.3.

Solubility

To determine the solubility of the various antibodies at roomtemperature, concentration experiments were performed using centrifugalfilter devices. Briefly, antibody preparations in PBS were added toVivaspin-15 (15 ml) centrifugal filter devices (30,000 MWCO, Sartorius,Goettingen, Germany) at room temperature. The filters were spun at3000×g for 20 minute intervals in a Beckman Allegra X15-R centrifugeusing a swinging bucket rotor. Once the volumes were reduced to about 2ml, the supernatant was transferred to a Vivaspin-4 (4 ml) filter device(30,000 MWCO) and centrifuged at 4,000×g for 20 min intervals. Once thevolume was reduced to 5001, the sample was transferred to a Vivaspin-500filter device and centrifuged at 15,000×g in an Eppendorf 5424centrifuge for 15 minutes. This was repeated until the proteinconcentration reached 100 mg/ml or more. The protein concentration wasdetermined by absorbance at 280 nm and 310 nm on a BioTek SynergyHT™spectrophotometer with appropriate dilution. At this point,centrifugation was stopped and the sample kept at room temperatureovernight to reach equilibrium. The next morning, the sample was checkedfor signs of precipitation. If the concentration was greater than 100mg/ml, the process was stopped.

Factor VIIA-Induced IL-8 Inhibition Assay

This assay was used to test whether or not the TF-binding antibodiesneutralize FVIIa-induced IL-8 release from human cells expressing TF.Human breast adenocarcinoma cells (MDA-MB-231) (ATCC: HTB-26), adaptedto grow in DMEM and 10% FBS (Gibco: cat #11995 and cat #16140), wereplated in 96-well cell culture plates (Nunc: cat #167008) at a densityof 20000 cells per well (100,000 cells/mL) using standard cell culturetechniques. The cells where allowed to recover for two days beforeantibody treatment starting at 2 ug/mL and undergoing either a 1:2 or1:4 serial dilution in DMEM without FBS. The antibody was added one hourbefore treatment with human FVIIa (Innovative Research: cat #IHFVIIa,lot: 2824) at a final concentration of 50 nM in DMEM without FBS. Thecells were placed in the incubator for 24 hours. After treatment, thesupernatant were collected and the quantity of IL-8 was detected byELISA according to manufactures protocol (R&D Systems: cat #D8000C).Briefly, the optical density (OD) of each treatment sample was read at450 nm and 540 nm. The reading at 540 nm was used to correct for opticalimperfection in the assay plate while the corrected reading at 450 nm(OD 450 minus OD 540) was used to calculate the IL-8 content in thetreatment samples using an IL-8 Standard Curve prepared according tomanufactures protocol. Wells with cells not receiving antibody and FVIIatreatment were used to define the endogenous IL-8 level while wells withcells only receiving FVIIa was used to define the “no inhibition” IL-8level, thereby defining the minimum and maximum IL-8 levels,respectively. The MAb titration treatment samples where normalized tothe maximum and minimum IL-8 levels as defined above and expressed aspercent inhibition. The normalized data were either represented in bargraphs or fitted to a four-parameter logistic curve fit to extract EC₅₀values for each MAb.

Coagulation Assay

This assay was used to determine whether or not the anti-human TFantibodies block coagulation in vitro using human plasma in the presenceof calcium and added recombinant human TF preparation (Innovin, DadeBehring Inc). Anti-human TF antibodies are diluted to 2 mg/ml antibodyin HB SS (Gibco, cat #14175). Pooled human plasma with Na Citrate(George King Biomedical, Novi, Mich.) is spun down at 1000 rpm for 5 minand clear plasma is transferred to a new tube. In each well of a clear96 well assay plate (NUNC, cat #439454), 25 ul of the diluted antibodyis added to 100 ul of the human plasma. The reaction is initiated byadding 125 μl of Innovin (Dade Behring Inc., cat #B4212) diluted 1:500into HBSS with 22 mM CaCl₂ to each well containing plasma with or withantibody. The coagulation reaction is kinetically monitored at OD 405,immediately following reaction initiation and for 30 min at 37° C. usinga SpectraMax M2e reader (Molecular Devices, Sunnyvale, Calif.). T½ Maxis determined for each antibody using Softmax Pro Software as the timein seconds it takes to reach 50% of the maximum optical density. Thetime in seconds for samples was normalized to a reference on each plate,however, statistically there was no difference between samples listing150 sec and 200 sec which was the average time for samples withoutantibody, for 10H10, and all of the 10H10 derived and human adaptedvariants.

Example 1 Sequencing of 10H10

The murine antibody known as 10H10 generated at The Scripps ResearchInstitute in La Jolla, Calif. (U.S. Pat. No. 5,223,427, Morrisey et al.1988 Thromb Res. 52(3): 247-261) is produced by the hybridomaTF9.10H10-3.2.2. The sequences of the antibody from the 10H10 hybridomaclone, had not been previously reported.

The sequences were identified using the 5′RACE method (Focus 25(2):25-27, 2003; Maruyama 1994 Gene 138, 171-174) where the two antibodychains, VH and VL, were amplified using 5′ GeneRacer™ (InVitrogen),primer and 3′ consensus primer complementary to a sequence within themouse IgG1 constant region and mouse Kappa constant region,respectively. Nested PCR amplification using 5′GeneRacer Nested primerand a 3′ consensus primer was used to generate VL products more suitablefor sequence analyses.

At least 16 clones were selected to identify the variable region of eachchain. Primers were used to sequence through the unknown region of theinserts. The raw sequence data was downloaded from the ABI DNA Sequencerto Vector NTI (Invitrogen Informax) for sequence analysis. Onefunctional VH and one functional VL were identified. Both VH and VLgenes were further analyzed to find their native signal sequences, FR,CDR, and J-segments.

The 10H10 FRs and CDRs are numbered sequentially, and segmentedfollowing Kabat's definition (Kabat et al., 5th edit. Public HealthService, NIH, Washington, D.C., 1991), except in the regioncorresponding to the CDR-1 of VH. For this region a combination of Kabatand Chothia definition was used (Raghunathan, G., US2009/0118127 A1;Chothia and Lesk, J Mol Biol 196(4): 901-17, 1987).

TABLE 110H10 Sequence of the Variable Regions and its Sequence StructuresProtein Name Polypeptide Sequence Subdomains 10H10 Heavy ChainQVHLQQSGAELMKPGASVKISCKAS FR1 = 1 to 25 Variable Region GYTFITYWIECDR1 = 26 to 35 (SEQ ID NO: 6) (SEQ ID NO: 4) WVKQRPGHGLEWIG FR2 =36 to 49 DILPGSGSTNYNENFKG CDR2 = 50 to 66 (SEQ ID NO: 7)KATFTADSSSNTAYMQLSSLTSEDSAVYYCAR FR3 = 67 to 98 SGYYGNSGFAY CDR3 =99 to 109 (SEQ ID NO: 8) WGQGTLVTVSA FR4(JH3) = 110 to 12010H10 Light Chain DIVMTQSPSSLTVTAGEKVTMSC FR 1 = 1 to 23 Variable RegionKSSQSLLSSGNQKNYLT CDR1 = 24 to 40 (SEQ ID NO: 9) (SEQ ID NO: 5)WYQQIPGQPPKLLIY FR2 = 41 to 55 WASTRES CDR2 = 56 to 62 (SEQ ID NO: 10)GVPDRFTGSGSGTDFTLTINSVQAEDLAVYYC FR3 = 63 to 94 QNDYTYPLT CDR3 =95 to 103 (SEQ ID NO: 11) FGAGTKLELK FR4(K5) = 104 to 113

The cloned V regions were engineered with human IgG1/Kappa constantregions and cloned into mammalian expression vectors for recombinantexpression in HEK293 or CHO cell lines creating a mouse-human chimericantibody designated M1, which was used in assay development as areference antibody. The HC V-region was also engineered with human IgG1CH1 domain only and a C-terminal hexahistidine for the purposes ofproducing a 10H10Fab used in crystal structure analysis.

Example 2 Epitope Mapping of a Non-Anticoagulant Tissue Factor Antibody

Epitope mapping for 10H10 was performed by crystal structuredetermination of a complex between human TF ECD and the correspondingFab fragment. The His-tagged human TF ECD (residues 5-213 of SEQ IDNO: 1) was expressed in Escherichia coli and purified by affinity andion exchange chromatography using a HisTrap HP column (GE Healthcare)and a Q HP column (GE Healthcare), respectively. The His-tagged chimericversions (mouse V regions, human constant domains) of 10H10 Fab wasexpressed in HEK cells and purified using affinity (a TALON column, GEHealthcare) and size exclusion (a HiLoad Superdex 200 column, GEHealthcare) chromatography.

The complex was prepared by mixing the Fab with human TF ECD at themolar ratio 1:1.2 (excess TF). The mixture was incubated for 20 min atroom temperature and loaded on a Superdex 200 column (GE Healthcare)equilibrated with 20 mM HEPES, pH 7.5, and 0.1 M NaCl. Fractionscorresponding to the main peak were pooled, concentrated to 10 mg/mL,and used for crystallization. The complex was crystallized by thevapor-diffusion method at 20° C. The 10H10:TF complex was crystallizedfrom solution containing 18% PEG 8000 in 0.1 M CHES, pH 9.5. For X-raydata collection, one crystal of the complex was soaked for a few secondsin the mother liquor supplemented with 20% glycerol, and flash frozen inthe stream of nitrogen at 100 K. X-ray diffraction intensities weremeasured using a Rigaku MicroMax™-007HF microfocus X-ray generatorequipped with a Saturn 944 CCD detector and an X-stream™ 2000cryo-cooling system (Rigaku). The structure was determined by molecularreplacement using the CCP4 suite of programs for macromolecularcrystallography (Collaborative Computational Project, Number 4. 1994.Acta Cryst. D50, 760-763).

The TF ECD consists of two topologically identical domains with theimmunoglobulin fold. The N-terminal domain spans residues 1-103, and theC-terminal domain spans residues 104-210 (of the ECD, SEQ ID NO: 1). The10H10 epitope was found to be centered on residues K149-D150 of the ECD,which reaches a deep pocket between the variable domains of the heavyand light chains of 10H10. The interface between 10H10 and TF isextensive and involves all six CDR loops (FIG. 1).

Notable findings are that the TF epitope of 10H10 does not overlap withthe FVII and FX binding sites (FIGS. 2 and 3). Also, the epitopes of10H10 and 5G9 (another murine human-TF binding antibody with ability toblock coagulation and which epitope was previously published, Huang etal. 1998 J Mol Biol 275:873-94) do partially overlap explaining thecompetitive binding between these two antibodies to human TF (FIGS. 2and 3).

Human TF ECD:10H10 Interface

10H10 binds TF at the interface between the N- and C-terminal domains ofthe ECD. The convex surface of TF fits to the concave CDR surface of theantibody. The total area buried upon complex formation is over 1,100 A2on each of the interacting molecules. All six CDRs are involved indirect contacts with TF (contacts defined as a 4-A interatomicdistance). In total, there are 24 epitope residues and 25 paratoperesidues. CDRs L1, H1 and H3 form the majority of contacts. Residuesforming the epitope and the paratope of the 10H10:TF complex are shownschematically in FIG. 1.

The 10H10 epitope includes two segments from the N-domain and threesegments from the C-domain of TF ECD. The two segments from the N-domaininteract with the antibody: residues 65-70 interacts with H-CDR1 andH-CDR3 and residue 104 interacting with H-CDR1. The three segments inthe C-domain interact with the antibody: residues 195 and 197 interactwith H-CDR1 and H-CDR2, residues 171-174 interact with L-CDR1 andL-CDR3, and residues 129-150 interact with L-CDR1, L-CDR3, H-CDR1 andH-CDR3; TF residues K149-D150 are in the center of the epitope; reachinga deep pocket formed between the VL and VH domains where their primarypartners are D97 of the LC variable region (SEQ ID NO:5) and W33 of theHC variable region (SEQ ID NO: 4) of 10H10, respectively.

B. Antibody Specificity

The amino acid sequences of human, cynomolgus (cyno) monkey (SEQ ID NO:2) and mouse TF ECD (SEQ ID NO: 3) are aligned in FIG. 2. There is highsimilarity between the sequences of human and cynomolgus TF ECD, and thetwo are only one residue different in the 10H10 contact residues:position 197 of SEQ ID NO:1, which is R (Arg) in the cyno sequence. Asthe T197 in the human sequence is contacted by a single H-CDR2 residue,the high level of cross-reactivity seen for the present set of 10H10derived antibodies is explainable.

By aligning the human and mouse TF sequences, the epitope residuesdetermining the species specificity of 10H10 were evident as significantamino acid differences occur in epitope residues: among the 24 residues10H10 contacts in human TF, the human and mouse sequences differ atpositions 68, 69, 70, and 104; in the N-domain and positions 136, 142,145 and 197 in the C-domain of SEQ ID NO: 1 versus SEQ ID NO: 3. Thedifferences are consistent with the diminished binding affinity of 10H10for mouse TF.

FIG. 2 also indicates the interaction sites on human TF for FVII and FXbased on a theoretical 3D model (FIG. 3) that describes theirassociation into a ternary complex (Norledge et al., Proteins53:640-648, 2003). Antibody 5G9 binds TF at an epitope that partiallyoverlaps with the FX binding site. Therefore 5G9 competes with FX andthis causes blockage of the coagulation cascade. 10H10 differs from 5G9in that it does not block coagulation while it effectively shuts off thesignaling via TF-associated PARs. Based on the model of the ternaryTF/FVII/FX complex, it was expected that the 10H10 epitope would be onthe free surface of TF and centered around residues K149-D150. Bindingof 10H10 mapped by mutagenesis and peptide epitope mapping providedearly evidence that this was the case.

The present crystal structure of the complex between TF ECD and 10H10Fab provides a spatial mapping of the manner in which the antibody canbind TF without preventing FVII and FX interaction either with TF oreach other. The 10H10 epitope as revealed by the structure covers thefree surface of TF as it theoretically exists in the ternary complex.Further, the 10H10 epitope partially overlaps that of the coagulationblocking MAb, 5G9, epitope (Huang 1998 supra), the common residues beingK149 and N171. Neither 10H10 or 5G9 block FVII binding to TF. Theepitopes of 10H10 and FX are also non-overlapping, but a steric clashoccurs between the constant domain of the Fab and the protease(globular) domain of FX in the current model. It should be noted howeverthat the orientation of FX may in fact differ from the model and thatthe association between FX and TF may allow some flexibility in theprotease domain. There is also a considerable flexibility in the elbowangle between the variable and constant domains of the Fab that mayallow to avoid the clash upon 10H10 binding to the ternary complex.

Example 3 Adapting the Binding Domains for Use in Humans

Efficacy of a therapeutic protein can be limited by unwanted immunereactions. Non-human monoclonal antibodies can have substantialstretches of linear amino acid sequences and local structuralconformations that can elicit immune response in humans. The transfer ofthe residues responsible for immunospecifity of target binding of anon-human MAb to a human antibody scaffold more often than not resultsin a substantial loss of binding affinity for the target antigen. Hence,it is highly valuable to use sound design principles for creatingantibody molecules that elicit minimal immunogenic reactions whileretaining the binding and biophysical profiles of the parent non-humanmolecule when injected into humans.

As previously described in US20090118127A1 and exemplified in Franssonet al. 2010 J Mol Biol 398:214-231, a two-step process was used to bothhumanize and restore or enhance binding affinity to generate theantibody species of the present invention which exhibit the targeteffects of the murine antibody 10H10 when engaging human TF. Thetwo-step process, called human framework adaption (HFA), consists of 1)human framework selection and 2) an affinity maturation step.

In the HFA process, the binding site residues (CDR) are combined withhuman germline genes selected based on sequence similarity andstructural considerations. The two systems of CDR assignments forantibodies are: the Kabat definition which is based upon antibodysequence variability and the Chothia definition based on analyses ofthree-dimensional structures of antibodies. Among the six CDRs, one orthe other system may be used where they diverge. In the case of thelight chain CDRs, the Kabat definition is used.

In the case of heavy chain CDR3, both Kabat and Chothia's definition areidentical. In the case of heavy chain CDR1, the Chothia definition wasused to define the start and the Kabat definition as the end (patterndefined by W followed by a hydrophobic amino acid such as V, I or A). Inthe case of VH-CDR2, Kabat's definition was used. However, in mostantibody structures, this sequence-based definition assigns a portion ofFR3 as belonging to CDR2. Thus, a shorter version of this CDR, whichends seven (7) residues earlier on the C-terminal region of this CDRcould also be used, called Kabat-7 herein.

Human FR Selection

Human FR's, defined as the regions in the V regions not comprised in theantigen-binding site, were selected from the repertoire of functionalhuman germline IGHV and IGHJ genes. The repertoire of human germlinegene sequences was obtained by searching the IMGT database (Lefranc2005) and compiling all “01” alleles. From this compilation, redundantgenes (100% identical at aminoacid level) and those with unpairedcysteine residues were removed from the compilation. The last update ofthe gene compilation was done on Oct. 1, 2007.

Initial selection of human sequences for HFA of VH was based on sequencesimilarity of the human VH germline genes to the entire length of themouse VH region including FR-1 to 3 as well as H-CDR-1 and H-CDR-2. Inthe next stage, the selected human sequences were rank ordered using ascore that takes into account both the length of the CDRs and sequencesimilarities between CDRs of mouse and human sequences. A standardmutation matrix, such as the BLOSUM 62 substitution matrix (Henikoff andHenikoff 1992 Proc Natl Acad Sci USA 15; 89(22):10915-9) was used forscoring alignments of the CDR's of mouse and human sequences and a largepenalty was applied if there was an insertion and/or deletion in the CDRloops. Human FR-4 was selected based on sequence similarity of the IGHJgermline genes (Lefranc 2005) with mouse 10H10 sequence, IGHJ4 (SEQ IDNO: 60).

A similar procedure was used for selecting human FRs for VL. In thiscase IGVK, germline genes selected using the same procedure than thatused for IGHV genes, served as genes for selecting FR's 1-3 and L-CDR1-3. Human IGJ-K2 gene (SEQ ID NO: 61) was selected as FR4 for allvariants.

Eleven VH and seven VL germline chains were selected. The VH genesselected were predominantly from IGVH-1 gene family: 6 sequences fromIGVH1 with IGVH1-69 and IGVH1-f used with longer and shorter H-CDR2, 2from IGVH3, and one IGVH5 gene used with both long and short H-CDR2. TheVL genes represented six of the IGVK2 and one of the IGVK4 gene family.

Thus, VH variants H15, H19 and H21 having the longer H-CDR2 (SEQ ID NO:7) correspond to H22, H23 and H24, respectively, where the shorter mouseCDR-H2 (SEQ ID NO: 27) was used. The prefix “s” denotes test variantswith fewer mouse residues and more human residues in the beta strandregion. The V-region designation used and the gene sequences used areshown in Tables 2 and 3 below.

TABLE 2 Polypetide ID IMGT Gene Used H13 VH-10H10 H14 IGHV1-2 H15IGHV5-a H16 IGHV1-46 H17 IGHV1-3 H18 IGHV3-74 H19 IGHV1-69 H20 IGHV1-18H21 IGHV1-f H22 s1_IGHV5-a H23 s1_IGHV1-69 H24 s1_IGHV1-f

TABLE 3 Peptide ID IMGT Gene Name L1 VL-10H10 L2 IGKV4-1_B3 L3IGKV2D40_O1 L4 IGKV2D-28_A3 L5 IGKV2D-29_A2 L6 IGKV2-30_A17 L7IGKV2-24_A232 L8 IGKV2D-26_A21

A library of 96 Mabs, representing the 11 heavy chain and 7 light chainhuman FR variants plus the murine 10H10 chimeric chains was expressed inHEK 293E cells in a 96-well format to provide supernatants for theprimary screen. For primary screening, using standard recombinantmethods, DNA encoding the selected variable domains was recombined toform complete MAbs which were transiently expressed in 96-well plates inHEK 293E cells. Supernatant fluid from the cultures was tested foractivity (binding) 96 hours following transfection.

Nineteen variants were chosen for pilot-scale expression in HEK 293-Fcells and purification based on the results of the primary screen.Pilot-scale expression was done transiently in HEK 293F cells or CHO-Sat a volume of 750 ml. The harvested supernatants were purified viaProtein A chromatography and the purified proteins were assessed fortheir affinity and functional activity. In addition, the purifiedproteins were subjected to biophysical characterization by SDS-PAGE,SE-HPLC, and cross-interaction chromatography (CIC). Theoreticalisoelectric points (pI) were calculated for each variant as well. Fromthe pilot scale characterizations, a set of final lead candidates wastransfected in WAVE bioreactors and purified via Protein Achromatography,

Binding Assessment

Binding of the parent chimeric antibody, M1, and HFA variants to bothhuman and cyno TF was performed as a direct ELISA format usingchemiluminescent detection. For primary screening of library variants incrude supernatants, samples or controls were normalized to 50 ng/ml inspent FreeStyle 293 HEK media (Gibco) and assayed at singleconcentration determinations. In the present assay, the concentration ofantibody was 5 ng (0.1 ml used) and the TF ECD and the antigen wasHis6-TF-ECD₁₋₂₁₉ used at a final concentration of 10 ng/well.

The results of screening of the entire combinatorial library showedthat, except H14, all other VHs bind to hTF with varying strengths.There are several HFA variants that gave a higher binding signal thanthe parent 10H10 (H13, L1), particularly some L3 and L5 combinations.H18 and H21 do not bind to human antigen as well as other VHs and showedinsignificant binding to cyno antigen. Among the VLs, L6 and L8 did notbind either antigen while others bound at detectable levels. H14 and L8also produced low expression when combined with any VL. There were 50 ofthe 77 antibody (VH, VL combinations) demonstrating TF binding as shownin Table 4.

TABLE 4 VH and VL Sequence IDs for the 50 Human TF Binding Human FRVariants Light Chain Light Chain Heavy Chain Heavy Chain Antibody IDPeptide ID SEQ ID NO: Peptide ID SEQ ID NO: M1 L1 5 H13 4 M9 L2 22 H1512 M10 L3 23 H15 12 M11 L4 24 H15 12 M12 L5 25 H15 12 M14 L7 26 H15 12M16 L2 22 H16 13 M17 L3 23 H16 13 M18 L4 24 H16 13 M19 L5 25 H16 13 M21L7 26 H16 13 M23 L2 22 H17 14 M24 L3 23 H17 14 M25 L4 24 H17 14 M26 L525 H17 14 M28 L7 26 H17 14 M30 L2 22 H18 15 M31 L3 23 H18 15 M32 L4 24H18 15 M33 L5 25 H18 15 M35 L7 26 H18 15 M37 L2 22 H19 16 M38 L3 23 H1916 M39 L4 24 H19 16 M40 L5 25 H19 16 M42 L7 26 H19 16 M44 L2 22 H20 17M45 L3 23 H20 17 M46 L4 24 H20 17 M47 L5 25 H20 17 M49 L7 26 H20 17 M51L2 22 H21 18 M52 L3 23 H21 18 M53 L4 24 H21 18 M54 L5 25 H21 18 M56 L726 H21 18 M58 L2 22 H22 19 M59 L3 23 H22 19 M60 L4 24 H22 19 M61 L5 25H22 19 M63 L7 26 H22 19 M65 L2 22 H23 20 M66 L3 23 H23 20 M67 L4 24 H2320 M68 L5 25 H23 20 M70 L7 26 H23 20 M72 L2 22 H24 21 M73 L3 23 H24 21M74 L4 24 H24 21 M75 L5 25 H24 21 M77 L7 26 H24 21

Based on relative binding affinity for TF using an ELISA, ten variantswere selected for scale up of expression and purification. A summary ofK_(D)s as measured by BIAcore, ELISA assay data, whole cell binding,inhibition of IL-8 induction from MDA-MB231 cells by 50 nM FVIIa by 2ug/ml Mab, and Tms as measured by the Thermoflour assay are shown inTable 5.

TABLE 5 Cyno Whole IL-8 Biacore Hu TF TF Cell Induction, K_(D) (nM) EC50EC50 Binding, Ave % MAb VH VL Human Cyno (ng/ml) (ng/ml) Ave %inhibition Tm ° C. M1 H13 L1 0.56 1.34 11.71 17.33 101% 106% 74.18 M10H15 L3 0.77 1.57 10.47 19.63  98% 104% 75.01 M11 H15 L4 0.37 1.52 9.9418.69 114%  93% 75.86 M12 H15 L5 0.55 2.24 10.05 22.8 124% 102% 79.72M16 H16 L2 0.66 1.96 9.31 23.39 109% 109% 78.56 M19 H16 L5 0.41 2.819.22 29.61 111% 106% 77.58 M58 H22 L2 0.2 1.18 9.03 19.81  94% 106%82.18 M59 H22 L3 0.4 1.28 8.8 18.67  95% 103% 75.94 M60 H22 L4 0.47 1.318.35 17.79 101% 106% 76.77 M61 H22 L5 0.21 1.71 8.3 24.07 111% 112%81.16 M9 H15 L2 0.61 1.55 9.8 19.93 107% 106% 79.43

Several of the new human MAb variants exhibited higher affinity for TFthan M1 (having the 10H10 variable chains: H13 and L1) and some werelower. The K_(D) for M61 (0.21 nM) is 2.5 times lower than that of themurine parent MAb (K_(D)=0.56 nM). The data in Table 5 includes fourMabs comprising H15 and four with H22 both constructed from the samegermline gene (IGHV5-a). Those with H22, and the shorter H-CDR2,generally exhibited higher binding affinity than the correspondingmolecules with H15. While many of the new variants bound cyno TF, theranking of cyno binding affinity differed from that for binding to humanTF.

The mouse 10H10 MAb Tm was 74.2° C. The Tm of the selected moleculesranged of Tms from 75 to 82.2° C. Thus, the HFA process resulted increation of antibody constructs with new Fd regions having increasedbinding affinity for human and nonhuman primate TF and also producedstable complete antibody variants with human domains.

Additional characterization of the new antibody constructs verified thatthe antibodies were capable of recognizing native TF on cellsoriginating from human tumor tissue (MDA-MB231 breast cancer derivedcells), and reduced TF signaling in the presence of VIIa as measured bythe suppressed induction of IL-8 from MDA-MB231.

Additional biophysical characterization (solubility andcross-interaction chromatography) and assay results led to the selectionof M59, comprised of the variable regions H22 and L3, for affinitymaturation.

Example 4 Antibody Maturation

The Fab libraries were constructed in a pIX phage display system asdescribed in U.S. Pat. No. 6,472,147 (Scripps) and applicants co-pendingapplication published as WO2009/085462 with minor modifications torestriction enzyme sites.

Based on the experimental structure of 10H10 in complex with hTF, twolibraries were designed starting with the M59 pairing of H116 (SEQ IDNO: 19) and L3 (SEQ ID NO: 23) for diversification of both V_(L) andV_(H). The libraries differed in terms of the positions targeted fordiversification, as well as in the amino acids used to diversifytargeted positions. One library diversified a total of eight of thepositions representing each CDR, previously shown to be in contact withTF. The emphasis in the design was put on L1, L3, H1 and H2. Positionsin contact in L2 were not diversified nor most of the positions in H3.Labile or reactive amino acids such as Cys and Met were avoided.

A second set of libraries was designed to diversify amino acids at theperiphery of the antigen-binding site determined by computing thesolvent accessibilities of the bound and unbound Fab crystal structure.Residues buried upon binding but also in contact with solvent moleculeswere targeted for diversification. A total of 12 residues (6 in V_(L)and 6 in VH) were identified using this method, which were diversifiedwith a reduced set of eight amino acids, including: Arg (R), Asn (N),Asp (D), Gly (G), His (H), Ser (S), Trp (W), and Tyr (Y). The size ofthe combined libraries was estimated to 8¹² or 10¹⁰ variants, which canbe covered using standard library restriction cloning techniques.

For the CDR contact residue library, the positions were diversified with15 amino acids (all except Met, Cys, Lys, Gln, and Glu) using anucleotide dimer (N-dimer) synthetic approach.

Fab libraries displayed on phage coat protein IX were panned againstbiotinylated hT-ECD according to panning schemes, known in the art,directed to increasing affinity by selecting for a slower off-rate(increase in off-rate value) or faster on-rate (decrease in on-ratevalue), or both were used. Panning used both human and cyno TF as targetantigen. Phage was produced by helper phage infection. Binders wereretrieved by addition of SA-beads to form a bead/antigen/phage complex.After the final wash, phage was rescued by infection of exponentiallygrowing TG-1 Escherichia coli cells. Phage was again produced andsubjected for additional rounds of panning.

The pIX gene was excised by NheI/SpeI digestion from the selected clonesand, after religation, DNA was transformed into TG-1 cells and grown onLB/Agar plates overnight. The overnight cultures were used for (i)colony PCR and sequencing for the V-regions, and (ii) soluble Fabproduction. The soluble Fab proteins were captured onto plates by apolyclonal anti-Fd(CH1) antibody. After appropriate washing andblocking, biotinylated hTF was added at 0.2 nM concentration andbiotinylated hTF was detected by HRP-conjugated streptavidin andchemiluminescence read as before. At this concentration of hTF, rankingof the Fab variants, defined as percent binding of the parent, in whichthe parent Fab, present as a control in all plates is defined as 100%binding, is possible.

By this criterion, 381 Fabs binding human TF at 100% or higher relativeto M59 Fab were selected.

A analysis of the selected clones, indicated certain changes at Y2, I5,T6 and Y7 of heavy chain CDR1 (SEQ ID NO:6) corresponding to positions27 and 30-32 of the V-region (SEQ ID NO:4 or 19) were successful.Although not a contact residue, position 27 only permitted an aromaticamino acid (Tyr and Phe). For positions 30-32, which have direct contactwith K68, T101, Y103 and L104 of human TF SEQ ID NO: 1 in the 10H10 Fab(FIG. 1), position 30 was relatively permissive but 31 and 32 wererestricted (Table 6).

In L-CDR2 changes in L3, S6 and S8 (SEQ ID NO: 7) corresponding topositions L52, S55 and S57 of SEQ ID NO: 4 or 19, which are residuesthat have direct contact with P194, 5195, and T197 of human TF SEQ IDNO:1 in the 10H10 Fab (FIG. 1) there was a somewhat restricted set ofsubstitutions permitted.

In H-CDR3, the N6 position (SEQ ID NO: 8) corresponding to N104 of SEQID NO: 4 or 19, which makes direct contact with F147, G148 and K149 ofhuman TF-ED (SEQ ID NO:1) appeared to be restricted.

The allowed amino acid changes in each of the library positions selectedby phage panning and screening for comparable binding affinity in solidphase capture assay using 0.2 nM human TF-ECD1-219 are shown in Table 6.

TABLE 6 HC_CDR1 HC_CDR2 HC_CDR3 Contact No Yes Yes Yes Yes No Yes No YesNo Yes No with Antigen in Position Y27 I30 T31 Y32 L52 G54 S55 G56 S57N59 N104 S105 in HC (SEQ ID NO: 19) Sequence Y I T Y L G S G S N N SDiversity F V P H I W T N Y V S A G A F V A V S W T N S H P L F L A V RA I T Y H H R F W S A L D P R Y F A D H

For VL in the selected clones, changes in S9, G10, N11 and K13 of L-CDR1(SEQ ID NO: 9) corresponding to positions 32, 33, 34 and 36 of the LCvariable region (SEQ ID NO: 5 or 23), which were shown by epitopemapping to contact E174, D129, S142, R144 and D145 of human TF SEQ IDNO:1 (FIG. 1) are shown in Table 7 as relatively permissive. For L-CDR2,residue W1, position 56 in SEQ ID NO: 5 or 23 (Kabat residue number 50),is a contact residue while residue E61 showed limited tolerability forsubstitution. In the light chain CDR3 (SEQ ID NO: 9) corresponding toresidue positions 97-100 of SEQ ID NO: 5 or 23, which have directcontact with K149, D150, N171 and T172 of human TF SEQ ID NO:1 (FIG. 1),position D97 (Kabat position 91) was restricted. The selection based onaffinity binding to human TF-ECD1-219 clone permissive amino acid usageis summarized in Table 7.

TABLE 7 LC-CDR1 LC-CDR2 LC-CDR3 Contact with Yes Yes Yes Yes Yes Yes NoYes Yes Yes Yes Antigen Position in LC S31 S32 G33 N34 K36 W56 E61 D97Y98 T99 Y100 (SEQ ID NO: 23) Sequence A A A A A A A D A A F Diversity DF F F H G D D D H F G G G I H E F F I G H H I L W G G G L H I I L N H HH N I L L N P N I L W L N N P R P L N Y N P P R S S N R P R R S V T S SS S S T V T T T T T V Y V V V V V Y Y Y W W W Y Y Y

In summary, a series of affinity improved Fab variants were identifiedthrough construction of a phage library based on directed variegation ofCDR positions of amino acid in the contact positions with antigen humanTF for the variable domains H22 (SEQ ID NO: 19) and L3 (SEQ ID NO: 23),followed by panning and selection of species with affinity in thescreening assay of comparable or better binding at compared to thestarting sequences.

Overall, modest increases in affinity for the VH and VL pairing selectedfrom the designed HFA libraries were obtained. However, from the VHlibrary where contact residues were variegated, the parent amino acidwas re-selected after stringent phage panning. Only two paratopepositions were significantly changed, as explained from structuralanalysis. These two amino acid replacements introduced in the CDRsduring affinity maturation, T31P and S57F, involve contact residues. The5-fold affinity improvement may be attributed to the increased interfaceof F57 which is in contact with S195 of TF. The VL interaction with TFseems to be plastic, allowing many changes to contact residues as wellas neighboring residues.

Of the 381 VH and VL pairings, 43 were selected for furthercharacterization. The selected 43 MAbs represented 27 different VHs and8 VLs (see Table 8 for the pairing of the heavy chain and light chainsequences) and could be classified into three sub-groups: Group 1variants have the same light chain (L3, SEQ ID NO: 23), and Group 2 andGroup 3 are represented by eight light chains paired with two differentheavy chains (H116 and H171, SEQ ID NO: 131 and 67, respectively).

TABLE 8 LC SEQ HC SEQ MAb ID LC ID ID NO: HC ID ID NO: M1583 L3 23 H177129 M1584 L3 23 H173 130 M1585 L3 23 H139 131 M1586 L3 23 H164 132 M1587L3 23 H116 133 M1588 L3 23 H179 134 M1589 L3 23 H187 135 M1590 L3 23H117 136 M1591 L3 23 H122 137 M1592 L3 23 H165 138 M1593 L3 23 H171 139M1594 L3 23 H158 140 M1595 L3 23 H160 141 M1596 L3 23 H185 142 M1597 L323 H134 143 M1598 L3 23 H137 144 M1599 L3 23 H130 145 M1601 L3 23 H105146 M1602 L3 23 H106 147 M1604 L3 23 H138 148 M1605 L3 23 H168 149 M1606L3 23 H181 150 M1607 L3 23 H189 151 M1610 L3 23 H115 153 M1611 L3 23H128 154 M1612 L3 23 H133 155 M1613 L3 23 H136 156 M1638 L138 157 H22 19M1639 L320 158 H22 19 M1640 L327 159 H22 19 M1641 L335 160 H22 19 M1642L369 161 H22 19 M1643 L162 162 H22 19 M1644 L225 163 H22 19 M1645 L283164 H22 19 M1646 L138 157 H171 139 M1647 L320 158 H171 139 M1648 L327159 H171 139 M1649 L335 160 H171 139 M1650 L369 161 H171 139 M1651 L162162 H171 139 M1652 L225 163 H171 139 M1653 L283 164 H171 139

Of the group of 27 antibodies having the same light chain as M59 (L3,SEQ ID NO: 23), the 27 heavy chains differ at three positions in H-CDR1(GYTFX₁X₂X₃WIE (SEQ ID NO: 83) where X1 is selected from A, D, G, I, L,N, P, R, S, T, V, Y and X2 is selected from A, P, S, and T and X3 isselected from F, H, and Y); except in H189 where H-CDR1 is GFTFITYWIA(SEQ ID NO: 81), and at four positions in H-CDR2 (DIX₁PGX₂GX₃TX₄ (SEQ IDNO: 107) where X1 is selected from 1 and L, X2 is selected from S and T,X3 is selected from A, F, H, and w; and X4 is selected from D, H, I, L,and N; except in H189 where H-CDR2 is DILPASSSTN (SEQ ID NO: 105)) whilethe H-CDR3 and FRs were unaltered from the H22 sequence (SEQ ID NO: 19)and is SGYYGNSGFAY (SEQ ID NO: 8). The unique compositions of the heavychains for these 27 Mabs are given below (Table 9).

TABLE 9  SEQ ID NO: SEQ ID NO: LC SEQ HC HC SEQ for for MAb  ID NO: ID ID NO: HC-CDR 1 H-CDR1 HC-CDR 2 H-CDR2 M1583 23 H177 129 GYTFGPYWIE 82DIIPGSGWTN 100 M1584 23 H173 130 GYTFVTYWIE 77 DILPGTGYTV 99 M1585 23H139 131 GYTFSPFWIE 70 DIIPGTGYTN 93 M1586 23 H164 132 GYTFPTYWIE 73DIIPGTGWTN 95 M1587 23 H116 133 GYTFIPYWIE 63 DILPGSGFTT 86 M1588 23H179 134 GYTFGPFWIE 78 DILPGSGYTN 101 M1589 23 H187 135 GYTFGPHWIE 80DILPGTGYTN 104 M1590 23 H117 136 GYTFLPYWIE 64 DIIPGTGFTN 88 M1591 23H122 137 GYTFRPYWIE 65 DIIPGTGYTN 93 M1592 23 H165 138 GYTFSPHWIE 74DILPGSGYTI 96 M1593 23 H171 139 GYTFAPYWIE 67 DILPGTGFTT 98 M1594 23H158 140 GYTFPPYWIE 72 DILPGTGYTV 99 M1595 23 H160 141 GYTFYPYWIE 72DILPGTGFTN 94 M1596 23 H185 142 GYTFTPYWIE 68 DILPGSGHTT 103 M1597 23H134 143 GYTFSSYWIE 70 DILPGTGATH 90 M1598 23 H137 144 GYTFTPYWIE 68DILPGTGYTV 99 M1599 23 H130 145 GYTFGPYWIE 82 DILPGTGYTL 89 M1601 23H105 146 GYTFGPYWIE 82 DILPGTGYTV 99 M1602 23 H106 147 GYTFDAHWIE 62DILPGSGFTD 84 M1604 23 H138 148 GYTFAPYWIE 76 DILPGTGYTW 92 M1605 23H168 149 GYTFGTYWIE 75 DILPGTGHTT 97 M1606 23 H181 150 GYTFIPHWIE 79DILPGSGWTN 102 M1607 23 H189 151 GFTFITYWIA 81 DILPASSSTN 105 M1610 23H115 153 GYTFAPYWIE 76 DIIPGTGYTT 85 M1611 23 H128 154 GYTFGPYWIE 82DILPGSGYTT 88 M1612 23 H133 155 GYTFNPYWIE 66 DILPGTGYTN 104 M1613 23H136 156 GYTFSSHWIE 69 DILPGSGFTH 91

H171 (SEQ ID NO: 139) comprises an additional change in H-CDR1 andH-CDR2 as compared to H116, which are 131A and S55T.

Two groups of Mabs are represented by eight LC (Table 10) paired withone of two different HC: H22 (SEQ ID NO: 19) or heavy chain H171 (SEQ IDNO: 139). The eight light chains all have the FR of L3 (derived fromIGKV240_O1) and have sequence changes in five positions in L-CDR1(KSSQSLLX₁X₂X₃X₄QX₅NYLT (SEQ ID NO: 116) where X1 is selected from F, P,S, T, W, and Y; X2 is selected from F, S, T, R, and V; X3 is selectedfrom A, G, P, S, W, Y, AND V; X4 is selected from G, N, and T; X5 isselected from K, R, and S), two in L-CDR 2 (X₁ASTRX₂S (SEQ ID NO: 120)where X1 is selected from H and W; X2 is selected from D, E and S) andfour in L-CDR3 (QNDX₁X₂X₃PX₄T (SEQ ID NO: 128) where X1 is selected fromD, F, and L; X2 is selected from S, T, and Y; where X3 is selected fromW, and Y; X4 is selected from L, and M). The compositions of the eightLC are shown in Table 10.

TABLE 10 LC SEQ ID NO: SEQ ID NO: SEQ ID NO: LC SEQ for L-CDR1 forL-CDR2 for L-CDR3 ID ID NO: L-CDR1 Sequence L-CDR2 Sequence L-CDR3SEQUENCE L138 156 108 KSSQSLLWFV 117 HASTRES 122 QNDDSYPL NQKNYLT T L162161 109 KSSQSLLYVY  10 WASTRES 121 QNDFSWPL GQKNYLT T L225 162 110KSSQSLLFRP  10 WASTRES 122 QNDDSYPL TQKNYLT T L283 163 111 KSSQSLLYTS 10 WASTRES 123 QNDDYWPL NQKNYLT T L320 157 112 KSSQSLLYSG 118 WASTRSS124 QNDDTYPM NQRNYLT T L327 158 113 KSSQSLLPSW  10 WASTRES 125 QNDFTYPLNQSNYLT T L335 159 114 KSSQSLLFSA 119 WASTRDS 126 QNDDTYPL NQRNYLT TL369 160 115 KSSQSLLTSY  10 WASTRES 127 QNDLTYPL NQRNYLT T

Some of these Mabs were subjected to further characterization and testedin in vivo xenograft models (Example 5).

Example 5 Characterization of Mabs

Following the human framework adaptation and reselection of a library ofvariants based on M59 comprising a single LC variable region (L3, SEQ IDNO. 23) and single HC variable region (H22, SEQ ID NO: 19), with alteredresidues in some CDR residues, the novel Mabs were subjected tobiophysical and bioactivity assays, and one paring, M1587, with alteredparatope residues was used to re-examine whether the epitope originallycharacterized for a 10H10 Fab binding to TF-ECD (Example 2) was altered.

Human TF ECD: M1587 Interface

The co-crystallization of human-adapted and affinity matured antibodybased on 10H10 CDRs, M1587 (L3 and H116) with TF ECD was performed inthe same manner as for 10H10 (Example 3) except that the M1587-Fab:TFcomplex was crystallized from solution containing 16% PEG 3350, 0.2 Mammonium acetate, 0.1 M sodium acetate, pH 4.5.

Comparison of the co-crystal structure of human TF ECD with that withthe affinity matured M1587 Fab indicates that the human adaptation andaffinity maturation of 10H10 has not changed the antibody epitopefootprint as shown in FIG. 2, nor have the conformation of the CDRs beenaltered. Three amino acid replacements (T31P, S57F, and N59T) wereintroduced in H-CDR1 and H-CDR2 (SEQ ID NO: 6 and 27, respectively,using the CDR definitions described in Example 2) during human frameworkadaption and affinity maturation (SEQ ID NO: 6 and 7 were replaced withSEQ ID NO: 63 and 86) including the contact residues at residue 31 and57 of H116 (SEQ ID NO: 133) which are T31P and S57F. There was afive-fold affinity improvement that may be attributed to the increasedinterface of F57, which is in contact with 5195 of TF. The structure ofhuman TF ECD with M1587 Fab confirmed the preservation of the epitopeduring HFA and affinity maturation even though changes were made in theH-CDR1 and H-CDR2 paratope residues.

Biophysical and Biological Assay Results

Summary data for these antibodies is show below for the KD analysis byBiacore (Table 11), coagulation time of human plasma by human TF (Table12), and the EC50 for IL8 release from MD-MB-231 cells stimulated withFVIIa (Table 13, 14, and FIG. 5).

The K_(D) for 43 selected affinity matured Mabs and includes datagenerated for the murine 10H10 and chimeric version of that MAb (M1)with selected human framework adapted variants with unmodified CDR from10H10 (M numbers less than 100) are shown in Table 11. Thus, thecombination of human framework selection and CDR residue substitutionproduced human antibodies with K_(D) in the range of 80 to 950 μM withan off rate (Koff) in the range of 2.2×10⁻⁵ sec-1 to 2.6×10⁻³ sec-1; andwith an on rate (Kon) in the range of 10⁴ M-1 sec-1 to 2.3×10⁵ M-1sec-1. Compared to values for the original murine 10H10 or the chimericconstruct, the novel Mabs have up to a 10-fold lower in equilibriumdissociation constant (K_(D)), from 0.77 nM to 0.08 nM; display a fasteron rate (K_(on)>10⁵ M-1 sec-1), or have a slower off rate (Koff=10⁵sec-1). These properties can be used to advantage in selecting a MAb forparticular applications where either residence time or ability topenetrate tissues is desired.

TABLE 11 ka kd Antibody (1/Ms) (1/s) K_(D) ID 10⁴ 10⁻⁴ (nM) M1639 4.693.71 0.08 M1645 5.21 4.12 0.08 M1647 3.87 4.02 0.1 M1652 4.44 4.82 0.11M1641 4.56 5.46 0.12 M1644 5.81 7.19 0.12 M1587 15.7 0.23 0.14 M160415.2 0.22 0.14 M1653 2.31 3.76 0.16 M1649 3.85 6.46 0.17 M1593 13.8 0.250.18 M1606 16.55 0.29 0.18 M1643 4.52 8.13 0.18 M1646 3.5 6.47 0.19M1650 2.33 4.5 0.19 M1651 2.01 3.88 0.19 M58 9.48 0.18 0.19 M1638 4.899.92 0.2 M61 9.41 0.19 0.2 M1584 18.4 0.56 0.3 M1611 19.2 0.59 0.31M1596 13.1 0.43 0.33 M1598 19.2 0.63 0.33 M1601 13.7 0.46 0.33 M158817.3 0.6 0.35 M1594 12.4 0.43 0.35 M1607 17 0.6 0.35 M11 9.73 0.35 0.36M1612 16.5 0.63 0.38 M1595 17.6 0.68 0.39 M1599 13.8 0.53 0.39 M158917.4 0.7 0.4 M1592 19.1 0.77 0.4 M1591 13.4 0.56 0.41 M19 9.88 0.41 0.41M59 9.33 0.38 0.41 M1583 23.9 1 0.42 M60 9.75 0.46 0.47 M1585 18.5 0.90.49 M46 9 0.44 0.49 M1610 18.4 1 0.54 M37 6.9 0.37 0.54 M12 9.22 0.510.55 M9 9.73 0.59 0.61 M1602 13.2 0.82 0.62 M1605 13.5 0.86 0.64 M1610.2 0.67 0.66 M1590 15.9 1.09 0.69 M1648 1.02 7.45 0.73 M1640 1.02 7.80.76 10H10 8.88 0.68 0.76 M1 9.52 0.74 0.77 M10 9.26 0.71 0.77 M158618.4 1.43 0.78 M1597 8.34 0.66 0.8 M1613 7.57 0.7 0.92 M1642 2.74 26.10.95 M42 8.04 0.82 1.02 M26 8.26 0.93 1.13 M68 6.43 1.03 1.6 M51 8.112.35 2.9 M52 7.4 2.64 3.57

Coagulation

The novel Mabs are characterized by the ability to bind human TF withoutblocking coagulation of human plasma as measured in vitro in thepresence of calcium and exogenously added human TF (Table 12). SeventeenHFA (M number less than 100) variants and 38 affinity maturated variants(M1583 and above) were assayed and the T_(1/2), Max (the time in secondsto reach 50% of the maximum optical density) reported.

All demonstrate responses similar to that observed with 10H10 (Table 12)with T_(1/2), Max values less than 205 seconds, indicating theseantibodies do not prolong the coagulation time when compared to thevehicle control with no antibody which was 159±17 (n=14). CNTO860, ahuman TF binding antibody described previously (U.S. Pat. No. 7,605,235B2) and derived from the murine antibody 5G9 which blocks FX binding toTF, prolongs clotting and never reaches coagulation within 1800 sec inthe same assay. Five of the 43 MAbs described in Example 4 as havingaltered CDRs, were not tested in the coagulation assay because they havestarting concentration less than 2 mg/ml. M1, M59 and CNTO860 valueswere averaged over multiple tests.

TABLE 12 T½ Max (seconds) SD MAb ID (n = 2) (seconds) 10H10* 191 2 M1*173 5 CNTO860* >900 11 M9 198 3 M10 196 6 M11 195 5 M12 197 7 M16 198 5M19 199 5 M26 186 4 M37 196 5 M42 202 7 M46 195 6 M51 207 7 M52 201 6M58 192 5 M59* 169 4 M60 180 4 M61 186 10 M68 207 11 M1583 189 8 M1584199 6 M1585 205 12 M1586 200 7 M1587 202 10 M1588 207 10 M1589 202 3M1590 180 5 M1591 154 6 M1592 163 6 M1593 164 7 M1594 165 5 M1595 163 5M1596 163 5 M1597 157 6 M1598 155 4 M1599 151 6 M1601 180 6 M1602 163 7M1604 165 5 M1605 160 5 M1606 157 5 M1607 152 6 M1610 164 4 M1611 154 10M1612 163 11 M1613 165 11 M1638 163 12 M1639 167 5 M1640 168 9 M1641 1629 M1642 166 6 M1643 148 6 M1644 150 6 M1645 154 4 M1647 160 6 M1648 1613 M1650 150 6

Signal Blocking Activity

The novel MAbs can also be described in terms of their ability to blocksignaling through the TF/FVIIa complex. TF/VIIa/PAR2 signaling of breastcancer cells induces a broad repertoire of proangiogenic factors such asVEGF25, Cyr61, VEGF-C, CTGF, CXCL1, and IL-8. It was previously reportedthat FVIIa induces detectable IL-8 in MDA-MB-231???, a human breastcancer cell line expressing TF (Albrektsen et al., J Thromb Haemost 5:1588-1597, 2007). Therefore, this assay was used as a biological assayto evaluate the variant antibodies ability to inhibit TF/VIIa inductionof IL-8 production.

The details of the assay are given herein above and the results oftesting 19 of the HFA (M10-M68) variants of Example 3 and 29 of the CDRvariants of Example 4 were tested for the ability to inhibit IL-8production at a single concentration of TF (0.5 microgm per ml). Ananti-RSV antibody (B37) that does not bind Tissue Factor, used as anegative control. At this concentration, many of the HFA Mabs were ableto block IL-8 induction by more than 67% (Table 13). FIG. 5 shows therelative inhibition of IL8-release by 27 of the MAbs sharing the L3light chain (SEQ ID NO: 23) and having substitutions in H-CDR1 or H-CDR2as compared to those of 10H10 (SEQ ID NO: 6 and 7, respectively). Inaddition; four of these: M1584, M1611, TF7M1612 and TF7M1607 were placedin a full titration IL-8 induction assay along with M. The relative IC50values calculated further support the observation that the affinityimproved variants are more potent as compared to M1, the mouse-humanchimera of 10H10 (Table 14). The other affinity mature groups ofaffinity maturated antibodies described in Example 4 produced similarresults.

TABLE 13 Variants % IL-8 ID Inhibition SD 10H10 93.9 8.0 M1 96.1 7.6 M9102.7 4.5 M10 90.7 0.0 M11 87.9 9.4 M12 98.6 3.1 M16 98.3 6.2 M19 87.35.8 M26 79.1 1.3 M37 71.2 11.6 M42 86.0 20.1 M46 82.5 10.7 M51 71.5 9.4M52 67.7 4.0 M58 88.5 8.5 M59 83.8 8.0 M60 99.6 4.5 M61 106.8 4.9 M6889.8 11.1

TABLE 14 MAb ID IC50 (ug/ml) M1 0.527 M59 0.382 M1584 0.332 M1607 0.395M1611 0.398 M1612 0.413

Example 6 Antibody Antitumor Activity

Mouse Xenograft Model with MDA-MB-231

MDA-MB-231 human breast cancer cells were cultured in DMEM medium with10% FBS and 1% LNN, harvested at log phase by trypsinization, andresuspended in sterile serum-free DMEM media at 5×10⁷ cells/mL. Twentyfemale SCID Beige (C.B-17/IcrCrl-scid-bgBR) mice were obtained fromCharles River Laboratories and acclimated for 14 days prior toexperimentation. At approximately eight weeks of age, mice wereimplanted in the right axillary mammary fat pad with 2.5×10⁶ MDA-MB-231cells. When tumors were approximately 100 mm³ in size, mice werestratified by tumor size into treatment groups (N=10 per group).Intraperitoneal treatment with Dulbecco's Phosphate Buffered Saline(DPBS) or M1593 at 10 mg/kg of body weight, commenced on the day ofstratification, and continued once weekly for a total of six doses.Tumors and body weights were recorded once weekly. The study terminatedwhen the mean tumor volume of each group reached 1500 mm³ Statisticaltests applied were two-way repeated measures ANOVA (PRIZM 4.0,GraphPad).

In MDA-MB-231 xenograft model, M1593 significantly inhibited tumorgrowth beginning on Day 22 (*P<0.01) and continuing until Day 29(**P<0.001), at which point the control (DPBS-treated) group waseuthanized. The M1593 treated group was euthanized on Day 36. M1593inhibited tumor growth by approximately 49% on Day 29. There was anapproximately 11-day tumor growth delay in the M1593 treated group,relative to the DPBS-treated control group (FIG. 6).

Mouse Xenograft Model with A431

A431 human squamous cell carcinoma cells were cultured in DMEM mediumwith 10% FBS and 1% LNN, harvested at log phase by trypsinization, andresuspended in sterile HBSS at 1×10⁷ cells/mL. Twenty female SCID Beige(C.B-17/IcrCrl-scid-bgBR) mice were obtained from Charles RiverLaboratories and acclimated for 14 days prior to experimentation. Atapproximately eight weeks of age, mice were implanted in the right flankwith 2×10⁶ A431 cells. When tumors were approximately 118 mm³ in size,mice were stratified by tumor size into treatment groups (N=10 pergroup). Intraperitoneal treatment with DPBS or M1593 at 10 mg/kg of bodyweight, commenced on the day of stratification, and continued onceweekly for a total of six doses. Tumors and body weights were recordedtwice weekly. The study terminated when the mean tumor volume of eachgroup reached 1000 mm³ Statistical tests applied were two-way repeatedmeasures ANOVA (PRIZM 4.0, GraphPad).

M1593 significantly inhibited tumor growth on Day 22 (*P=0.0067), atwhich point the control (DPBS treated) group was euthanized. TheCNTO592-treated group was euthanized on Day 39. CNTO592 inhibited tumorgrowth by approximately 54% on Day 22. There was an approximately 17-daytumor growth delay in the M1593 treated group, relative to theDPBS-treated control group (FIG. 7).

Example 7 Antibody Compositions with Altered F_(C)

Naturally occurring human Fc receptor variants have substantiallydiffering affinities for the Fc-portion of human antibodies. Inaddition, clinical studies have demonstrated improved response rates andsurvival for patients with tight binding Fc genotypes after treatmentwith Fc-engineered mAbs (Musolino et al 2008 J Clin Oncol 26:1789-1796(2008); Bibeau et al 2009 J Clin Oncol 27:1122-1129).

While inhibition of TF signaling is expected to reduce cellularresponses leading to tumor proliferation, migration, and metastasicspread, the fact that TF antigen is displayed on tumor cells providesfor a means for selective killing of the target cell by mechanismsrelated to Fc-receptor engagement by the antibody Fc. The surfacefeatures of the Fc-domain of the antibody are known to be influenced bythe glycan composition as well as the primary sequence of the heavychains, and modification of either or both can alter Fc-receptorbinding.

The MAb identified as M1593, was produced as a low fucoseglycan-modified IgG1 and also as a IgG1-CH2 domain variant (S239D, 1332Ewhere the numbering is that of Kabat EU system).

MAb Compositions and Methods of Making

The antibody with low fucose content (M1593-LF) was produced byelectroporating a vector encoding the M1593 (IgG1/Kappa) chains as shownbelow with signal peptides into a CHO host cell subline selected for lowfucosylation of proteins from the CHO host cell line. SEQ ID NO: 165represents the complete light chain comprising the variable domain,residues 1-113 (SEQ ID NO: 23 plus FR4, SEQ ID NO: 61, underlined) andthe human kappa constant light domain. The heavy chains comprising thevariable domain residues 1-120 (which includes SEQ ID NO: 139 and FR4SEQ ID NO: 60, underlined) with wild-type human IgG isotype1 CH1, CH2,and CH3 where the Kabat positions 239 and 332 (which are 242 And 335 ofSEQ ID NO: 167) are modified from the wild-type residues S and D, to Dand E, respectively to form the variant M1593-DE.

M1593-Light Chain (SEQ ID NO: 165)DIVMTQTPLSLPVTPGEPASISCKSSQSLLSSGNQKNYLTWYLQKPGQSPQLLIYWASTRESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQNDYTYPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECM1593-heavy chain where Kabat position S239 is D, and I332 is E(SEQ ID NO: 167) EVQLVQSGAEVKKPGESLRISCKGSGYTFAPYWIEWVRQMPGKGLEWMGDILPGTGFTTYSPSFQGHVTISADKSISTAYLQWSSLKASDTAMYYCARSGYYGNSGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGP D VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP E EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K

The CHO cell was generated by sub-cloning after four rounds of negativelectin selection (selection of non-binding to fucose binding lectin) andFACS sorting to isolate a pool of naturally occurring low fucose cellsto be used as a host cell line. This line was derived from the same hostcells used to produced M1593 and therefore, the cells are cultured andhandled exactly in the same manner. Transfected cells were screened bymethylcellulose plating using protein G for detection and coloniespicked into 96-well plates. Cultures were expanded to shaker flasks fortitering. Top parental clones produced M1593-LF up to 708 mg/L (instandard medium) in batch shake flask cultures.

LC-MS glycopeptide mapping was performed on two M1593-LF producingclones (C2452B and C2452D) to determine the percent fucosylation andevaluate the stability of the glycosylation profile over time andproduction process (Table 15). Samples were collected during a stabilitystudy from passage 1 fed-batch and passage 10 batch cultures andpurified. Purified samples from the bioreactor evaluation were alsoanalyzed. Glycopeptide mapping showed favorable glycosylation patternswith low percent total fucosylation from C2452B and C2452D. Importantly,the percent fucosylation did not significantly increase over time whichsuggests that the host cell fucosylation is stable. Thus, the fucosecontent for M1593-LF is less than 10%, and generally, less than 5% and,in some preparations, less than 2%. Mab produced in the non-lectinselected host CHO cell comprised glycan groups where greater than 80%were fucosylated.

TABLE 15 Clone % Fucosylation Sample Analyzed C2452B 2.81 P1 Shake FlaskFed-batch C2452B 1.83 p10 Batch of Stability Study C2452B 3.67Bioreactor C2452D 2.20 P1 Shake Flask Fed-batch C2452D 2.25 p10 Batch ofStability Study C2452D 7.31 Bioreactor

For the mutant Fc variant of M1593 (M1593-DE), the plasmid expressingM1593 was subjected to site directed mutagenesis.

Biological Activity

The three anti-human TF Fc variants (M1593, M1593-LF, and M1593-DE)affinity for both human and cynomolgus Fc receptors (FcγRI, FcγRIIa,FcγRIIIa). These assays were performed as described in applicantsco-pending application (U.S. Ser. No. 61/426,619) or by using Plasmonresonance (Biacore) based binding assays (?).

The results of these assays demonstrated that both Fc modified anti-TFantibodies bound much more tightly to recombinant human FcγIIIareceptors compared with the parental, unmodified IgG1 M1593 antibody by18-fold (M1593-LF) and 40-fold (M1593-DE) (Table 16).

TABLE 16 Anti-TF antibody affinities for human FcγIIIa receptors MAbHuman Fcγ111a K_(D) (M) M1593 (wild-type human IgG1/kappa 2.1 × 10⁻⁷constant domains) M1593-DE (hIgG1 with S239D/I332E) 5.0 × 10⁻⁹ M1593-LF(hIgG1 produced in host subline 1.2 × 10⁻⁸ producing reducedfucosylation in glycans)

ADCC is stimulated by FcγRIIIa engagement. ADCC assays were performed aspreviously described (Scallon et al., Mol Immunol 44:1524-1534 2007).

Improved Fc receptor binding was reflected in functional in vitro ADCCassays using human PBMC as effector cells and human breast cancer cellline MDA-MB-231 as the target cell (FIG. 8).

What is claimed:
 1. An isolated antibody which competes for binding tohuman tissue factor with the murine antibody 10H10, wherein the antibodybinding domains are adapted to a human framework (FR) region, and havingthe structure of FR1-CDR1-FR2-CDR2-FR3-CDR3 wherein the FR amino acidsequences are unaltered from the amino acid sequences encoded by humangermline gene sequences where the germline is recognized in the IMGTdatabase and the CDR sequences bear not less than 50% sequence identityto the murine 10H10 CDRs represented by SEQ ID NO: 6-11 and
 27. 2. Theisolated antibody of claim 1, wherein the antibody does not compete withFVIIa for tissue factor binding and does not substantially block theprocoagulant, amidolytic activity of the TF-VIIa complex but which doesblock TF-VIIa mediated signaling as measured by cytokine IL-8 releasefrom MDA-MB-231 cells.
 3. The antibody of claim 2, wherein one or moreof the CDR sequences are selected from sequences of SEQ ID NO: 6-11 and27.
 4. The antibody of claim 3, wherein the three light chain CDRsequences, L-CDR1, L-CDR2 and L-CDR3 are represented by SEQ ID NO: 9-11,respectively.
 5. The antibody of claim 3, wherein the three heavy chainCDR sequences, H-CDR1, H-CDR2 and H-CDR3 are represented by SEQ ID NO:6-8, respectively or by SEQ ID NO: 6, 27 and 8, respectively.
 6. Theantibody of claim 3, wherein L-CDR1, L-CDR2 and L-CDR3 are representedby SEQ ID NO: 9-11, respectively, and the three heavy chain CDRsequences, H-CDR1, H-CDR2 and H-CDR3 are represented by SEQ ID NO: 6-8,respectively or by SEQ ID NO: 6, 27 and 8, respectively.
 7. The antibodyof claim 1, wherein the human HC variable region frameworks are derivedfrom an IGHV family 1, 3 or 5 member as represented by the IMGTdatabase.
 8. The antibody of claim 7, comprising a HC variable regionselected from SEQ ID NO: 12-21.
 9. The antibody of claim 1, wherein thehuman LC variable region frameworks are derived from a human IGKV familytwo or four member.
 10. The antibody of claim 9, comprising a LCvariable region selected from SEQ ID NO: 22-26.
 11. The antibody ofclaim 1, wherein the human HC variable region frameworks are derivedfrom a human germline gene family selected from IGHV5 and IGKV2.
 12. Theantibody of claim 1, comprising a HC variable region selected from SEQID NO: 12-21 and a human LC variable region selected from SEQ ID NO:22-26.
 13. The antibody of claim 1, comprising a HC variable regionhaving the H-CDR3 of SEQ ID NO: 8; an H-CDR1 having a sequence selectedfrom SEQ ID NO: 6, 62-83; an H-CDR2 having a sequence selected from SEQID NO: 7, 27, and 84-107; and a HC FR4 region, optionally, selected fromIGVJ4 (SEQ ID NO: 60) or a variant thereof.
 14. The antibody of claim 1,comprising a LC variable region having an L-CDR1 having a sequenceselected from SEQ ID NO: 9, 108-116; an L-CDR2 having a sequenceselected from SEQ ID NO: 10 and 117-120; and an L-CDR3 having a sequenceselected from SEQ ID NO: 11 and 121-128; and a LC FR4 region,optionally, selected from IGKJ2 (SEQ ID NO: 61) or a variant thereof.15. The antibody of claim 1, comprising a HC variable region having theH-CDR3 of SEQ ID NO: 8; an H-CDR1 having a sequence selected from SEQ IDNO: 6, 62-83; an H-CDR2 having a sequence selected from SEQ ID NO: 7,27, and 84-107; and a HC FR4 region, optionally, selected from IGVJ4(SEQ ID NO: 60) or a variant thereof, and a LC variable domain having anL-CDR1 having a sequence selected from SEQ ID NO: 9, 108-116; an L-CDR2having a sequence selected from SEQ ID NO: 10 and 117-120; and an L-CDR3having a sequence selected from SEQ ID NO: 11 and 121-128; and a LC FR4region, optionally, selected from IGKJ2 (SEQ ID NO: 61) or a variantthereof.
 16. The antibody of claim 1, wherein the HC human frameworksequences are derived from IGHV5_a and the HC variable region has thesequence selected from SEQ ID NO: 19, 129-155.
 17. The antibody of claim1, wherein the LC human FR sequences are derived from IGKV2D40_O1 andthe LC variable region has the sequence selected from SEQ ID NO: 23,157-164.
 18. The antibody of claim 1, wherein the HC human frameworksequences are derived from IGHV5_a and the HC variable region has thesequence selected from SEQ ID NO: 19, 129-155, the LC human FR sequencesare derived from IGKV2D40_O1 and the LC variable domain has the sequenceselected from SEQ ID NO: 23, 157-164.
 19. The antibody of claim 1 havinga binding domain derived from IGHV5_a frameworks, defined as non-CDRpositions, an H-CDR3 having the sequence SGYYGNSGFAY (SEQ ID NO: 8) andwherein the sequences at the H-CDR-1 and/or H-CDR-2 positions are givenby the formulas: H-CDR1 (SEQ ID NO: 83) GYTFX₁X₂X₃WIE (I)

where X1 is selected from A, D, G, I, L, N, P, R, S, T, V, and Y; X2 isselected from A, P, S, and T and X3 is selected from F, H, and Y; or thesequence may be GFTFITYWIA (SEQ ID NO: 81); and H-CDR2 (SEQ ID NO: 107)DIX₁PGX₂GX₃TX₄ (II)

where X1 is selected from 1 and L, X2 is selected from S and T, X3 isselected from A, F, H, and w; and X4 is selected from D, H, I, L, and N;or where H-CDR2 is DILPASSSTN (SEQ ID NO: 105).
 20. The antibody ofclaim 1 having a binding domain wherein the non-CDR positions arederived from IGKV2D40_O1 frameworks and wherein the sequences at theL-CDR-1 and/or LCDR-2, and L-CDR3 have the sequences given by theformulas: L-CDR1 (SEQ ID NO: 116) KSSQSLLX₁X₂X₃ X₄Q X₅NYLT (III)

where X1 is selected from F, P, S, T, W, and Y; X2 is selected from F,S, T, R, and V; X3 is selected from A, G, P, S, W, Y, AND V; X4 isselected from G, N, and T; and X5 is selected from K, R, and S; L-CDR2(SEQ ID NO: 120) X₁ASTRX₂S (IV)

where X1 is selected from H and W; X2 is selected from D, E and S;L-CDR3 (SEQ ID NO: 128) QNDX₁X₂X₃PX₄T (V)

where X1 is selected from D, F, and L; X2 is selected from S, T, and Y;where X3 is selected from W, and Y; and X4 is selected from L, and M.21. A method of treating a human subject suffering from a condition inwhich TF-expression and local bioactivity resulting from theTF-expression is directly or indirectly related to the condition to betreated which comprises administering to such a subject in need of suchtreatment an antibody according to claim 1, 3 19 or
 20. 22. A methodaccording to claim 21 wherein the condition is cancer.
 23. A methodaccording to claim 22 wherein the cancer is selected from primary solidtumors, metastases; carcinomas, adenocarcinomas, melanomas, liquidtumors, lymphomas, leukemias, myelomas, soft tissue cancers, sarcomas,osteosarcoma, thymoma, lymphosarcoma, fibrosarcoma, leiomyosarcoma,lipomas, glioblastoma, astrosarcoma, cancer of the prostate, breast,ovary, stomach, pancreas, larynx, esophagus, testes, liver, parotid,biliary tract, colon, rectum, cervix, uterus, endometrium, thyroid,lung, kidney, or bladder.
 24. A method according to claim 21 wherein thecondition is selected from benign tumors, hemangiomas, acousticneuromas, neurofibromas, trachomas, and pyogenic granulomas;artheroscleric plaques; ocular angiogenic diseases, diabeticretinopathy, retinopathy of prematurity, macular degeneration, cornealgraft rejection, neovascular glaucoma, retrolental fibroplasia,rubeosis, retinoblastoma, uveitis and Pterygia (abnormal blood vesselgrowth) of the eye; rheumatoid arthritis; psoriasis; delayed woundhealing; endometriosis; vasculogenesis; granulations; hypertrophic scars(keloids); nonunion fractures; scleroderma; trachoma; vascularadhesions; myocardial angiogenesis; coronary collaterals; cerebralcollaterals; arteriovenous malformations; ischemic limb angiogenesis;Osler-Webber Syndrome; plaque neovascularization; telangiectasia;hemophiliac joints; angiofibroma; fibromuscular dysplasia; woundgranulation; Crohn's disease; and atherosclerosis
 25. A pharmaceuticalcomposition useful in treating a subject comprising an antibody of claim1, 3, 19 or 20 in a pharmaceutically acceptable preparation.
 26. A kitcomprising an antibody according to claim 1, 3, 19 or 20 in a stableform and instructions for use.
 27. An isolated nucleic acid encoding oneor more of the antibody binding domains of an antibody according toclaim 1, 3 19 or
 20. 28. A vector comprising at least one polynucleotideof claim
 27. 29. A host cell comprising the vector of claim
 28. 30. Theisolated antibody or fragment of claim 1, 3, 19 or 20, having an IgG1 orIgG4 isotype.
 31. The isolated antibody or fragment of claim 30, whereinthe Fc region comprises human IgG1 isotype where the Kabat positions 239and 332 (which are 242 And 335 of SEQ ID NO: 167) are modified from thewild-type residues S and D, to D and E mutations in the Fc region.